HUMAN-RNA HELICASE AND THERAPEUTIC USES THEREOF
BACKGROUND OF THE INVENTION
RNA helicases are enzymes that unwind or rearrange duplex and structured RNA molecules and are required at almost every discrete step of major RNA processing events. RNA helicases are also involved in mRNA degradation. RNA helicases of the DExH/D family not only can unwind duplex RNA but are reported to disrupt high-affinity RNA-protein interactions using the energy released by ATP hydrolysis, although the precise mechanism of action remains to be characterized.
The stability of many mRNAs is dynamically regulated both positively and negatively by a variety of signals acting on specific sequences in the RNA molecule. Among various cis-acting instability elements so far identified, the most conspicuous is the AU-rich element (ARE) located in the 3' untranslated region of unstable mRNAs. The presence of an ARE and its function as an instability determinant have been reported for many mRNAs with short half-lives including those encoding cytokines, growth factors and protooncoproteins. Proteins that bind to the ARE are suspected to act to increase or decrease the stability of ARE-mRNAs, although the mechanism of action is unknown. Disregulation of normal mRNA decay may contribute to pathological conditions; in neoplasia, increased ARE-mRNA stability is thought to be one of the factors contributing to this disease state.
We report herein the isolation and characterization of the human RNA helicase, RHAU (RNA helicase associated AU-rich element) as well as the identification, isolation and characterization of a heretofore unidentified alternatively spliced isoform of RHAU, referred to herein as RHAUΔ14. While the human gene for RHAU had previously been predicted and identified based on bioinformatic analysis (Strausberg RL et al., 2002, PNAS USA 99: 16899-16903; Nagase T et al., 2000, DNA Res. 7: 143-150; Fu JJ. et al., 2002, Acta Biochimica et Biophysica Sinica 34: 655-661), studies involving the cDNA of human RHAU, the actual isolation or further characterization of the human RHAU protein or the existence of RHAUΔ14have not yet been reported.
Functional characterization of the human RHAU protein reported herein reveals that not only can RHAU facilitate ARE-mRNA degradation in vitro and in vivo, data indicate that the downregulation of RHAU protein levels inhibits the growth of tumor cells, suggesting that RHAU is an important enzyme for cancer cell biology and may be helpful to facilitate the search, e.g., for specific small molecule inhibitors of tumor cell growth. Thus, it is postulated herein that RHAU can be used as a novel drug target useful in the diagnosis, prevention, and treatment of human diseases, particularly neoplasia. The invention provides methods for identifying modulators of RHAU activity and gene expression and the use of such modulators, e.g., for the treatment of cancer. The invention also provides pharmaceutical compositions comprising said modulators.
SUMMARY OF THE INVENTION
The instant invention relates to the identification and isolation of RNA helicase polypeptides, allelic variants and other mammalian orthologs thereof. These unique polypeptide sequences, and nucleic acid sequences that encode these peptides, can be used as models for the identification and development of therapeutic targets, aid in the identification of therapeutic proteins, and serve as targets for the development of therapeutic agents that modulate enzyme activity in cells and tissues that express the enzyme. DETAILED DESCRIPTION OF THE INVENTION
Peptide Molecules
In one aspect, the present invention provides amino acid sequences of previously uncharacterized human RNA helicases referred to herein as RHAU and RHAUΔ14, nucleic acid sequences in the form of transcript sequences, and cDNA sequences that encode RHAU peptides and proteins and variants thereof. As discussed above, the peptides that are provided in the present invention may be useful as drug targets.
The amino acid sequence of RHAU is provided herein as SEQ ID NO:3 and the amino acid sequence of an allelic variant missing 14 amino acids (amino acids 517-530), RHAUΔ14, is provided herein as SEQ ID NO:4. These amino acid sequences are referred to interchangeably herein as the enzyme peptides of the present invention, enzyme peptides, or peptides/proteins of the present invention. Unless otherwise apparent from context, the term "RHAU" as used herein is meant to include RHAUΔU. Nucleic acid sequences encoding the RHAU and RHAUΔ14 peptides are provided herein as SEQ ID NO:1 and SEQ ID NO:2, respectively. These sequences are deposited in GenBank under accession numbers AJ577133 and AJ577134. It is understood that the peptides of the present invention may be encoded by obvious allelic and functionally equivalent variants of the nucleic acid sequences provided herein.
As used herein, a peptide is said to be "isolated" or "purified" when it is substantially free of cellular material or free of chemical precursors or other chemicals. The peptides of the present invention can be purified to homogeneity or other degrees of purity. The level of purification will be based on the intended use. The critical feature is that the preparation allows for the desired function of the peptide, even if in the presence of considerable amounts of other components. In some uses, "substantially free of cellular material" includes preparations of the peptide having less than about 30% (by dry weight) other proteins (i.e., contaminating protein), less than about 20% other proteins, less than about 10% other proteins, or less than about 5% other proteins. When the peptide is recombinantly produced, it can also be substantially free of culture medium, i.e., culture medium represents less than about 20% of the volume of the protein preparation.
The language "substantially free of chemical precursors or other chemicals" includes preparations of the peptide in which it is separated from chemical precursors or other chemicals that are involved in its synthesis. In one embodiment, the language "substantially free of chemical precursors or other chemicals" includes preparations of the enzyme peptide having less than about 30% (by dry weight) chemical precursors or other chemicals, less than about 20% chemical precursors or other chemicals, less than about 10% chemical precursors or other chemicals, or less than about 5% chemical precursors or other chemicals.
The isolated enzyme peptide can be purified from cells that naturally express it, purified from cells that have been altered to express it (recombinant), or synthesized using known protein synthesis methods. For example, a nucleic acid molecule encoding the enzyme peptide may be cloned into an expression vector, the expression vector introduced into a host cell and the protein expressed in the host cell. The protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques.
Accordingly, the present invention provides proteins that may consist of, consist essentially of, or comprise the amino acid sequences provided in SEQ ID NO:3 and SEQ ID NO:4, for example, proteins encoded by the nucleic acid sequence of SEQ ID NO:1 and SEQ ID NO:2, respectively. Generally, a protein "consists of" an amino acid sequence when the amino acid sequence is the final amino acid sequence of the protein; a protein "consists essentially of" an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein; and a protein "comprises" an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterologous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids. The preferred classes of proteins that are comprised of the enzyme peptides of the present invention are the naturally occurring mature proteins.
The enzyme peptides of the present invention can be attached to heterologous sequences to form chimeric or fusion proteins. Such chimeric and fusion proteins comprise an enzyme peptide operatively linked to a heterologous protein having an amino acid sequence not substantially homologous to the enzyme peptide. Operatively linked" indicates that the enzyme peptide and the heterologous protein are fused in-frame. The heterologous protein can be fused to the N- terminus or C-terminus of the enzyme peptide.
In some uses, the fusion protein does not affect the activity of the enzyme peptide per se. For example, the fusion protein can include enzymatic fusion proteins, for example, beta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-His fusions, MYC-tagged, Hl-tagged or Ig fusions. Such fusion proteins, particularly poly-His fusions, can facilitate the purification of recombinant enzyme peptide. In certain host cells (e.g., mammalian host cells), expression and/or secretion of a protein can be increased by using a heterologous signal sequence. A chimeric or fusion protein can be produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different protein sequences are ligated together in-frame in accordance with conventional techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence. Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST protein). An enzyme peptide-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the enzyme peptide.
As mentioned above, the present invention also provides obvious variants of the amino acid sequence of the proteins of the present invention, such as naturally occurring mature forms of the peptide, allelic/sequence variants of the peptides, non-naturally occurring recombinantly derived variants of the peptides, and orthologs and paralogs of the peptides. Such variants can readily be generated using art-known techniques in the fields of recombinant nucleic acid technology and protein biochemistry.
Variants can readily be identified/made using molecular techniques and the sequence information disclosed herein. Further, such variants can readily be distinguished from other peptides based on sequence and/or structural homology to the enzyme peptides of the present invention. The degree of homology/identity present will be based primarily on whether the peptide is a functional variant or non-functional variant, the amount of divergence present in the paralog family and the evolutionary distance between the orthologs.
To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
The comparison of sequences and determination of percent identity and similarity between two sequences can be accomplished using a mathematical algorithm known to one of skill in the art. Such algorithms may be found in: Computational Molecular Biology, Lesk AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith D W, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1 , Griffin AM and Griffin HG, eds, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje G, Academic Press, 1987; and Sequence Analysis Primer, Gribskov M and Devereux J, eds, M Stockton Press, New York, 1991. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (J. MoI. Biol. 48: 444-453, 1970) which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (Devereux J et al., 1984, Nucleic Acids Res. 12: 387; available at http://www.gcg.com), using a NWSgapdna. CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of Myers and Miller (CABIOS 4: 11-17, 1989) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
The nucleic acid and protein sequences of the present invention can further be used as a "query sequence" to perform a search against sequence databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (J. MoI. Biol. 215: 403-410, 1990). Gapped BLAST can be utilized as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When utilizing BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.
Full-length pre-processed forms, as well as mature processed forms, of proteins that comprise one of the peptides of the present invention can readily be identified as having complete sequence identity to one of the enzyme peptides of the present invention as well as being encoded by the same genetic locus as the enzyme peptide provided herein.
Allelic variants of an enzyme peptide can readily be identified as being a human protein having a high degree of, or significant, sequence homology/identity to at least a portion of the enzyme peptide as well as being encoded by the same genetic locus as the enzyme peptide provided herein. As used herein, two proteins (or a region of the proteins) have a high degree of, or significant homology when the amino acid sequences are typically at least about 70-80%, 80-90%, and more typically at least about 90-95% or more homologous. A significantly homologous amino acid sequence, according to the present invention, will be encoded by a nucleic acid sequence that will hybridize to an enzyme peptide encoding nucleic acid molecule under stringent conditions as more fully described below.
Paralogs of an enzyme peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the enzyme peptide, as being encoded by a gene from humans, and as having similar activity or function. Two proteins will typically be considered paralogs when the amino acid sequences are typically at least about 60% or greater, and more typically at least about 70% or greater homology through a given region or domain. Such paralogs will be encoded by a nucleic acid sequence that will hybridize to an enzyme peptide encoding nucleic acid molecule under moderate to stringent conditions as more fully described below.
Orthologs of an enzyme peptide can readily be identified as having some degree of significant sequence homology/identity to at least a portion of the enzyme peptide as well as being encoded by a gene from another organism. Preferred orthologs will be isolated from mammals, preferably primates, for the development of human therapeutic targets and agents. Such orthologs will be encoded by a nucleic acid sequence that will hybridize to an enzyme peptide encoding nucleic acid molecule under moderate to stringent conditions, as more fully described below, depending on the degree of relatedness of the two organisms yielding the proteins.
Non-naturally occurring variants of the enzyme peptides of the present invention can readily be generated using recombinant techniques. Such variants include, but are not limited to deletions, additions and substitutions in the amino acid sequence of the enzyme peptide. For example, one class of substitutions are conserved amino acid substitution which involve the substitution of a given amino acid in an enzyme peptide by another amino acid of like characteristics. Such substitutions are familiar to one of skill in the art.
Variant enzyme peptides can be fully functional or can lack function in one or more activities, e.g. ability to bind substrate, ability to phosphorylate substrate, ability to mediate signaling, etc. Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids that result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree.
Non-functional variants typically contain one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncation or a substitution, insertion, inversion, or deletion in a critical residue or critical region.
Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis; mutant molecules may be tested for biological activity such as enzyme activity or in assays such as an in vitro proliferative activity. Sites that are critical for binding partner/substrate binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling techniques familiar to one of skill in the art.
The present invention further provides fragments of the enzyme peptides, in addition to proteins and peptides that comprise and consist of such fragments. As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or more contiguous amino acid residues from an enzyme peptide. Such fragments can be chosen based on the ability to retain one or more of the biological activities of the enzyme peptide or could be chosen for the ability to perform a function, e.g. bind a substrate or act as an immunogen. Particularly important fragments are biologically active fragments, peptides that are, for example, about 8 or more amino acids in length. Such fragments will typically comprise a domain or motif of the enzyme peptide, e.g., active site, a transmembrane domain or a substrate-binding domain. Further, possible fragments include, but are not limited to, domain or motif containing fragments, soluble peptide fragments and fragments containing immunogenic structures. Predicted domains and functional sites are readily identifiable by computer programs well known and readily available to those of skill in the art (e.g., PROSITE analysis).
Polypeptides often contain amino acids other than the 20 amino acids commonly referred to as the 20 naturally occurring amino acids. Further, many amino acids, including the terminal amino acids, may be modified by natural processes, such as processing and other post-translational modifications, or by chemical modification techniques well known in the art. Common modifications that occur naturally in enzyme peptides are described in basic texts, detailed monographs, and the research literature, and they are well known to those of skill in the art.
The enzyme peptides of the present invention also encompass derivatives or analogs in which a substituted amino acid residue is not one encoded by the genetic code, in which a substituent group is included, in which the mature enzyme peptide is fused with another compound, such as a compound to increase the half-life of the enzyme peptide (for example, polyethylene glycol), or in which the additional amino acids are fused to the mature enzyme peptide, such as a leader or secretory sequence or a sequence for purification of the mature enzyme peptide or a pro-protein sequence.
Protein/Peptide Uses
The proteins of the present invention can be used in various applications, e.g., to raise antibodies or to elicit an immune response; as a reagent (including as a labeled reagent) in assays designed to quantitatively determine levels of the protein (or its binding partner or ligand) in biological fluids; and as markers for tissues in which the corresponding protein is preferentially expressed (either constitutively or at a particular stage of tissue differentiation or development or in a disease state). Where the protein binds or potentially binds to another protein or ligand (such as, for example, in an enzyme-effector protein interaction or enzyme-ligand interaction), the protein can be used to identify the binding partner/ligand so as to develop a system to identify inhibitors of the binding interaction. Any of these uses are capable of being developed into reagent grade or kit format for commercialization as commercial products and the methods for performing the uses listed above are well known to those skilled in the art.
Enzymes isolated from humans and their human/mammalian orthologs may also serve as targets for identifying agents for use in mammalian therapeutic applications, e.g. a human drug, particularly in modulating a biological or pathological response in a cell or tissue that expresses the enzyme.
Experimental data as provided in Example 2 disclosed herein indicate that down regulation of levels of the RHAU helicase using RHAU-specific siRNA results in inhibition of the growth of tumor cells in vitro suggesting that this helicase may be a useful target for identifying antineoplastic agents.
As well as those discussed herein, additional uses may be readily be determined using the information provided herein, that which is known in the art, and routine experimentation.
In addition, the proteins of the present invention (including variants and fragments thereof) are useful for biological assays related to enzymes that are related to members of the helicase subfamily. Such assays may involve any of the known enzyme functions or activities or properties useful for diagnosis and treatment of enzyme-related conditions that are specific for the family of enzymes to which the RHAU proteins belong, particularly in cells and tissues that express the enzyme.
The proteins of the present invention are also useful in drug screening assays, in cell-based or cell-free systems. Cell-based systems can be native, i.e., utilizing cells that normally express the enzyme, as a biopsy or expanded in cell culture. In an alternate embodiment, cell-based assays involve recombinant host cells expressing the enzyme protein.
The polypeptides can be used to identify compounds that modulate enzyme activity of the protein in its natural state or an altered form that causes a specific disease or pathology associated with the enzyme. The enzymes of the present invention and appropriate variants and fragments can be used in high-throughput screens to assay candidate compounds for the ability to bind to the enzyme. These compounds can be further screened against a functional enzyme to determine the effect of the compound on the enzyme activity. Further, these compounds can be tested in animal or invertebrate systems to determine activity/effectiveness. Compounds can be identified that activate (agonist) or inactivate (antagonist) the enzyme to a desired degree.
Further, the proteins of the present invention can be used to screen a compound for the ability to stimulate or inhibit interaction between the enzyme protein and a molecule that normally interacts with the enzyme protein, e.g., a substrate or a component of the signal transduction pathway with which the enzyme protein normally interacts (for example, another enzyme). Such assays typically include the steps of combining the enzyme protein with a candidate compound under conditions that allow the enzyme protein, or fragment, to interact with the target molecule, and to detect the formation of a complex between the protein and the target or to detect the biochemical consequence of the interaction with the enzyme protein and the target, such as any of the associated effects of signal transduction such as protein phosphorylation, cAMP turnover and adenylate cyclase activation, etc. For example, ATPase activity of RHAU was analysed by detection of free pi on this layer chromatography (TLC) after reaction. After incubation of RHAU and [γ-32 P] ATP, reaction was spotted on TLC, then TLC was developed. Free 32P visualized by phosphor-imager. Candidate compounds may include, for example, peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries and combinatorial chemistry-derived molecular libraries made of D- and/or L-configuration amino acids; phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries; antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab')2, Fab expression library fragments, and epitope-binding fragments of antibodies); and small organic and inorganic molecules (e.g., molecules obtained from combinatorial, chemical and natural product libraries).
One candidate compound may be a soluble fragment of a receptor that competes for substrate binding. Other candidate compounds include mutant enzymes or appropriate fragments containing mutations that affect enzyme function and thus compete for substrate. Accordingly, a fragment that competes for substrate, for example with a higher affinity, or a fragment that binds substrate but does not allow release, is encompassed by the invention.
The invention further includes other end point assays to identify compounds that modulate (stimulate or inhibit) enzyme activity. The assays typically involve an assay of events in the signal transduction pathway that indicate enzyme activity. Thus, the phosphorylation of a substrate, activation of a protein, a change in the expression of genes that are up- or down-regulated in response to the enzyme protein dependent signal cascade can be assayed.
Any of the biological or biochemical functions mediated by the enzyme can be used as an endpoint assay. Specifically, a biological function of a cell or tissues that express the enzyme can be assayed.
Binding and/or activating compounds can also be screened by using chimeric enzyme proteins in which the amino terminal extracellular domain, or parts thereof, the entire transmembrane domain or subregions, such as any of the seven transmembrane segments or any of the intracellular or extracellular loops and the carboxy terminal intracellular domain, or parts thereof, can be replaced by heterologous domains or subregions. For example, a substrate-binding region can be used that interacts with a different substrate than that which is recognized by the native enzyme. Accordingly, a different set of signal transduction components is available as an end-point assay for activation. This allows for assays to be performed in cells other than the specific host cell from which the enzyme is derived.
The proteins of the present invention are also useful in competition binding assays in methods designed to discover compounds that interact with the enzyme (e.g. binding partners and/or ligands). For example, a compound is exposed to an enzyme polypeptide under conditions that allow the compound to bind or to otherwise interact with the polypeptide. Soluble enzyme polypeptide is also added to the mixture. If the test compound interacts with the soluble enzyme polypeptide, it decreases the amount of complex formed or activity from the enzyme target. This type of assay is particularly useful in cases in which compounds are sought that interact with specific regions of the enzyme. Thus, the soluble polypeptide that competes with the target enzyme region is designed to contain peptide sequences corresponding to the region of interest.
To perform cell free drug screening assays, it is sometimes desirable to immobilize either the enzyme protein, or fragment, or its target molecule to facilitate separation of complexes from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. In one embodiment, a fusion protein, e.g., a glutathione-S-transferase (GST) fusion protein can be created using methods familiar to one of skill in the art which provides a domain that allows the protein to be bound to a matrix. Alternatively, antibodies reactive with the protein but which do not interfere with binding of the protein to its target molecule can be derivatized to the wells of a plate, and the protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the enzyme protein target molecule, or which are reactive with enzyme protein and compete with the target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the target molecule.
Agents that modulate one of the enzymes of the present invention can be identified using one or more of the above assays, alone or in combination. It is generally preferable to use a cell-based or cell free system first and then confirm activity in an animal or other model system. Such model systems are well known in the art and can readily be employed in this context.
Modulators of enzyme protein activity identified according to these drug screening assays can be used to treat a subject with a disorder mediated by the enzyme pathway, by treating cells or tissues that express the enzyme. Experimental data as provided in the Examples herein indicate that such modulators of RHAU activity may be useful to treat neoplasia. Methods of treatment may include the steps of administering a modulator of enzyme activity in a pharmaceutical composition to a subject in need of such treatment, the modulator being identified as described herein. Pharmaceutical compositions are discussed in more detail below.
In yet another aspect of the invention, the enzyme proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay familiar to one of skill in the art to identify other proteins which bind to or interact with the enzyme and are involved in enzyme activity. Such enzyme-binding proteins are also likely to be involved in the propagation of signals by the enzyme proteins or enzyme targets as, for example, downstream elements of an enzyme-mediated signaling pathway. Alternatively, such enzyme-binding proteins may be enzyme inhibitors. This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., an enzyme-modulating agent, an antisense enzyme nucleic acid molecule, siRNA, ribozyme, an enzyme-specific antibody, or an enzyme-binding partner) can be used in an animal or other model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal or other model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
The enzyme proteins of the present invention are also useful to provide a target for diagnosing a disease or predisposition to disease mediated by the peptide. Accordingly, the invention provides methods for detecting the presence, or levels of, the protein (or encoding mRNA) in a cell, tissue, or organism. The method involves contacting a biological sample with a compound capable of interacting with the enzyme protein such that the interaction can be detected. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.
One agent for detecting a protein in a sample is an antibody capable of selectively binding to protein. A "biological sample" as used herein, is used in its broadest sense and may comprise tissues, cells and biological fluids isolated from a subject.
The peptides of the present invention also provide targets for diagnosing active protein activity, disease, or predisposition to disease, in a patient having a variant peptide, particularly activities and conditions that are known for other members of the family of proteins to which the present one belongs. Thus, the peptide can be isolated from a biological sample and assayed for the presence of a genetic mutation that results in aberrant peptide. This includes amino acid substitution, deletion, insertion, rearrangement, (as the result of aberrant splicing events), and inappropriate post-translational modification. Analytic methods include altered electrophoretic mobility, altered tryptic peptide digest, altered enzyme activity in cell-based or cell-free assay, alteration in substrate or antibody-binding pattern, altered isoelectric point, direct amino acid sequencing, and any other of the known assay techniques useful for detecting mutations in a protein. Such an assay can be provided in a single detection format or a multi-detection format such as an antibody chip array.
In vitro techniques for detection of peptides include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence using a detection reagent, such as an antibody or protein binding agent. Alternatively, the peptide can be detected in vivo in a subject by introducing into the subject a labeled anti-peptide antibody or other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. Particularly useful are methods that detect the allelic variant of a peptide expressed in a subject and methods which detect fragments of a peptide in a sample.
The peptides may also be subjects of pharmacogenomic analysis, which takes into consideration that the genotype of an individual can determine the way a therapeutic compound acts on the body or the way the body metabolizes the compound and suggests that the selection of effective compounds and effective dosages of such compounds for prophylactic or therapeutic treatment be based on the individual's genotype. Accordingly, therapeutically effective dosages could be discerned and modified as necessary to maximize the beneficial effect within a given population containing a particular polymorphism. The peptides are also useful for treating a disorder characterized by an absence of, inappropriate, or unwanted expression of the protein. Accordingly, methods for treatment include the use of the enzyme protein or fragments.
Antibodies
The invention also provides antibodies that selectively bind to the peptides of the present invention, a protein comprising such a peptide, as well as variants and fragments thereof. As used herein, an antibody selectively binds a target peptide when it binds the target peptide and does not significantly bind to unrelated proteins. An antibody is still considered to selectively bind a peptide even if it also binds to other proteins that are not substantially homologous with the target peptide so long as such proteins share homology with a fragment or domain of the peptide target of the antibody. In this case, it would be understood that antibody binding to the peptide is still selective despite some degree of cross- reactivity.
The antibodies of the present invention include, but are not limited to, humanized or chimeric antibodies, single chain antibodies, anti-idiotypic antibodies, polyclonal antibodies and monoclonal antibodies, as well as fragments of such antibodies, including, but not limited to, Fab or F(ab')2, and Fv fragments. These antibodies may be generated using conventional methods familiar to one of skill in the art.
Antibodies are preferably prepared from regions or discrete fragments of the enzyme proteins. Antibodies can be prepared from any region of the peptide as described herein. However, preferred regions will include those involved in function/activity and/or enzyme/binding partner interaction and include the conserved motifs responsible for the coordinated coupling between substrate binding, NTP binding/hydrolysis and unwinding activity. Methods for detecting antibodies to a given target peptide are also well known to one of skill in the art. Detection of an antibody of the present invention can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
Antibody Uses
The antibodies can be used to isolate the proteins of the present invention by standard techniques, such as affinity chromatography or immunoprecipitation. The antibodies can facilitate the purification of the natural protein from cells and recombinantly produced protein expressed in host cells. In addition, such antibodies are useful to detect the presence of the proteins of the present invention in cells or tissues to determine the pattern of expression of the protein among various tissues in an organism and over the course of normal development. Further, such antibodies can be used to detect protein in situ, in vitro, or in a cell lysate or supernatant in order to evaluate the abundance and pattern of expression. Also, such antibodies can be used to assess abnormal tissue distribution or abnormal expression during development or progression of a biological condition. Antibody detection of circulating fragments of the full length protein can be used to identify turnover.
Further, the antibodies can be used to assess expression in disease states such as in active stages of the disease or in an individual with a predisposition toward disease related to the protein's function. When a disorder is caused by an inappropriate tissue distribution, developmental expression, level of expression of the protein, or expressed/processed form, the antibody can be prepared against the normal protein. If a disorder is characterized by a specific mutation in the protein, antibodies specific for this mutant protein can be used to assay for the presence of the specific mutant protein. The antibodies can also be used to assess normal and aberrant subcellular localization of cells in the various tissues in an organism. The diagnostic uses can be applied, not only in genetic testing, but also in monitoring a treatment modality. Accordingly, where treatment is ultimately aimed at correcting expression level or the presence of aberrant sequence and aberrant tissue distribution or developmental expression, antibodies directed against the protein or relevant fragments can be used to monitor therapeutic efficacy.
Additionally, antibodies are useful in pharmacogenomic analysis. Thus, antibodies prepared against polymorphic proteins can be used to identify individuals that require modified treatment modalities. The antibodies are also useful as diagnostic tools as an immunological marker for aberrant protein analyzed by electrophoretic mobility, isoelectric point, tryptic peptide digest, and other physical assays known to those in the art.
The antibodies are also useful for tissue typing. Thus, where a specific protein has been correlated with expression in a specific tissue, antibodies that are specific for this protein can be used to identify a tissue type.
The antibodies are also useful for inhibiting protein function, for example, blocking the binding of the enzyme peptide to a binding partner such as a substrate. These uses can also be applied in a therapeutic context in which treatment involves inhibiting the protein's function. An antibody can be used, for example, to block binding, thus modulating (agonizing or antagonizing) the peptides activity. Antibodies can be prepared against specific fragments containing sites required for function or against intact protein that is associated with a cell or cell membrane.
The invention also encompasses kits for using antibodies to detect the presence of a protein in a biological sample. The kit can comprise antibodies such as a labeled or labelable antibody and a compound or agent for detecting protein in a biological sample; means for determining the amount of protein in the sample; means for comparing the amount of protein in the sample with a standard; and instructions for use. Such a kit can be supplied to detect a single protein or epitope or can be configured to detect one of a multitude of epitopes, such as in an antibody detection array using conventional methodologies.
Nucleic Acid Molecules
The present invention further provides isolated nucleic acid molecules that encode an enzyme peptide or protein of the present invention. Such nucleic acid molecules will consist of, consist essentially of, or comprise a nucleotide sequence that encodes one of the enzyme peptides of the present invention, an allelic variant thereof, or an ortholog or paralog thereof.
As used herein, an "isolated" nucleic acid molecule is one that is separated from other nucleic acid present in the natural source of the nucleic acid. Preferably, an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 51 and 31 ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. However, there can be some flanking nucleotide sequences, for example up to about 5KB, 4KB, 3KB, 2KB, or 1 KB or less, particularly contiguous peptide encoding sequences and peptide encoding sequences within the same gene but separated by introns in the genomic sequence. The important point is that the nucleic acid is isolated from remote and unimportant flanking sequences such that it can be subjected to the specific manipulations described herein such as recombinant expression, preparation of probes and primers, and other uses specific to the nucleic acid sequences.
Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or chemical precursors or other chemicals when chemically synthesized. However, the nucleic acid molecule can be fused to other coding or regulatory sequences and still be considered isolated. For example, recombinant DNA molecules contained in a vector are considered isolated. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the isolated DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
Accordingly, the present invention provides nucleic acid molecules that consist of, consist essentially of or comprise the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:2 or any nucleic acid molecule that encodes the proteins provided in SEQ ID NO:3 and SEQ ID NO:4. In general, a nucleic acid molecule consists of a nucleotide sequence when the nucleotide sequence is the complete nucleotide sequence of the nucleic acid molecule. A nucleic acid molecule consists essentially of a nucleotide sequence when such a nucleotide sequence is present with only a few additional nucleic acid residues in the final nucleic acid molecule. A nucleic acid molecule comprises a nucleotide sequence when the nucleotide sequence is at least part of the final nucleotide sequence of the nucleic acid molecule. In such a fashion, the nucleic acid molecule can be only the nucleotide sequence or have additional nucleic acid residues, such as nucleic acid residues that are naturally associated with it or heterologous nucleotide sequences. Such a nucleic acid molecule can have a few additional nucleotides or can comprise several hundred or more additional nucleotides.
The isolated nucleic acid molecules can encode the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature peptide (when the mature form has more than one peptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, facilitate protein trafficking, prolong or shorten protein half-life or facilitate manipulation of a protein for assay or production, among other things. As generally is the case in situ, the additional amino acids may be removed from the mature protein by cellular enzymes.
As mentioned above, the isolated nucleic acid molecules include, but are not limited to, the sequence encoding the enzyme peptide alone, the sequence encoding the mature peptide and additional coding sequences, such as a leader or secretory sequence (e.g., a pre-pro or pro-protein sequence), the sequence encoding the mature peptide, with or without the additional coding sequences, plus additional non-coding sequences, for example introns and non-coding 5' and 31 sequences such as transcribed but non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals), ribosome binding and stability of mRNA. In addition, the nucleic acid molecule may be fused to a marker sequence encoding, for example, a peptide that facilitates purification.
Isolated nucleic acid molecules can be in the form of RNA, such as mRNA, or in the form of DNA, including cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The nucleic acid, especially DNA, can be double-stranded or single-stranded. Single- stranded nucleic acid can be the coding strand (sense strand) or the non-coding strand (anti-sense strand).
The invention further provides nucleic acid molecules that encode fragments of the peptides of the present invention as well as nucleic acid molecules that encode obvious variants of the enzyme proteins of the present invention that are described above. Such nucleic acid molecules may be naturally occurring, such as allelic variants (same locus), paralogs (different locus), and orthologs (different organism), or may be constructed by recombinant DNA methods or by chemical synthesis. Such non-naturally occurring variants may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells, or organisms. Accordingly, as discussed above, the variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions.
The present invention further provides for the addition of non-coding fragments to the nucleic acid molecules provided in SEQ ID NO:1 and SEQ ID NO:2. Preferred non-coding fragments include, but are not limited to, promoter sequences, enhancer sequences, gene modulating sequences and gene termination sequences. Such fragments are useful in controlling heterologous gene expression and in developing screens to identify gene-modulating agents and methodologies for their manipulation are well known to one of skill in the art.
As referred to herein, a "fragment" comprises a contiguous nucleotide sequence greater than 12 or more nucleotides. Further, a fragment could be at least 30, 40, 50, 100, 250 or 500 nucleotides in length. The length of the fragment will be based on its intended use. For example, the fragment can encode epitope bearing regions of the peptide, or can be useful as DNA probes and primers. Such fragments can be isolated using the known nucleotide sequence to synthesize an oligonucleotide probe. A labeled probe can then be used to screen a cDNA library, genomic DNA library, or mRNA to isolate nucleic acid corresponding to the coding region. Further, primers can be used in PCR reactions to clone specific regions of gene.
A probe/primer typically comprises substantially a purified oligonucleotide or oligonucleotide pair. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, 20, 25, 40, 50 or more consecutive nucleotides.
Orthologs, homologs, and allelic variants can be identified using methods well known in the art. As described above, these variants comprise a nucleotide sequence encoding a peptide that is typically 60-70%, 70-80%, 80-90%, and more typically at least about 90-95% or more homologous to the nucleotide sequence disclosed herein or a fragment of this sequence. Such nucleic acid molecules can readily be identified as being able to hybridize under moderate to stringent conditions, to the nucleotide sequence shown herein or a fragment of the sequence. Allelic variants can readily be determined by genetic locus of the encoding gene.
As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences encoding a peptide at least 60-70% homologous to each other typically remain hybridized to each other. The conditions can be such that sequences at least about 60%, at least about 70%, or at least about 80% or more homologous to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1- 6.3.6. One example of stringent hybridization conditions are hybridization in 6 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 x SSC, 0.1% SDS at 50-65°C. Examples of moderate to low stringency hybridization conditions are also well known in the art.
Nucleic Acid Molecule Uses
The nucleic acid molecules of the present invention are useful for probes, primers, chemical intermediates, and in biological assays. The nucleic acid molecules are useful as a hybridization probe for messenger RNA, transcript/cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding the peptides described in SEQ ID NO:3 and SEQ ID NO:4 and to isolate cDNA and genomic clones that correspond to variants (alleles, orthologs, etc.) producing the same or related peptides disclosed herein.
The probe can correspond to any sequence along the entire length of the nucleic acid molecules provided herein. Accordingly, it could be derived from 5' non- coding regions, the coding region, and 3' non-coding regions.
The nucleic acid molecules are also useful as primers for PCR to amplify any given region of a nucleic acid molecule and are useful to synthesize antisense molecules of desired length and sequence.
The nucleic acid molecules are also useful for designing ribozymes corresponding to all, or a part, of the mRNA produced from the nucleic acid molecules described herein.
The nucleic acid molecules are also useful for constructing recombinant vectors. Such vectors include expression vectors that express a portion of, or all of, the peptide sequences. Vectors also include insertion vectors, used to integrate into another nucleic acid molecule sequence, such as into the cellular genome, to alter in situ expression of a gene and/or gene product. For example, an endogenous coding sequence can be replaced via homologous recombination with all or part of the coding region containing one or more specifically introduced mutations.
The nucleic acid molecules are also useful for expressing antigenic portions of the proteins.
The nucleic acid molecules are also useful as probes for determining the chromosomal positions of the nucleic acid molecules by means of in situ hybridization methods.
The nucleic acid molecules are also useful in making vectors containing the gene regulatory regions of the nucleic acid molecules of the present invention.
The nucleic acid molecules also provide vectors for gene therapy in patients containing cells that are aberrant in enzyme gene expression. Thus, recombinant cells, which include the patient's cells that have been engineered ex vivo and returned to the patient, are introduced into an individual where the cells produce the desired enzyme protein to treat the individual. Gene therapy methodologies are familiar to one of skill in the art.
The nucleic acid molecules are also useful for making vectors that express part, or all, of the peptides.
The nucleic acid molecules are also useful for constructing host cells expressing a part, or all, of the nucleic acid molecules and peptides.
The nucleic acid molecules are also useful for constructing transgenic animals expressing all, or a part, of the nucleic acid molecules and peptides.
The nucleic acid molecules are also useful as hybridization probes for determining the presence, level, form and distribution of nucleic acid expression. Accordingly, the probes can be used to detect the presence of, or to determine levels of, a specific nucleic acid molecule in cells, tissues, and in organisms. DNA or RNA levels may be determined. Accordingly, probes corresponding to the peptides described herein can be used to assess expression and/or gene copy number in a given cell, issue, or organism. These uses are relevant for diagnosis of disorders involving an increase or decrease in enzyme protein expression relative to normal results.
In vitro techniques for detection of mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detecting DNA include Southern hybridizations and in situ hybridization.
Probes can be used as a part of a diagnostic test kit for identifying cells or tissues that express an enzyme protein, such as by measuring a level of an enzyme-encoding nucleic acid in a sample of cells from a subject e.g., mRNA or genomic DNA, or determining if an enzyme gene has been mutated.
Nucleic acid expression assays are useful for drug screening to identify compounds that modulate enzyme nucleic acid expression.
The invention thus provides a method for identifying a compound that can be used to treat a disorder associated with nucleic acid expression of the enzyme gene, particularly biological and pathological processes that are mediated by the enzyme in cells and tissues that express it. The method typically includes assaying the ability of the compound to modulate the expression of the enzyme nucleic acid and thus identifying a compound that can be used to treat a disorder characterized by undesired enzyme nucleic acid expression. The assays can be performed in cell-based and cell-free systems. Cell-based assays include cells naturally expressing the enzyme nucleic acid or recombinant cells genetically engineered to express specific nucleic acid sequences.
The assay for enzyme nucleic acid expression can involve direct assay of nucleic acid levels, such as mRNA levels, or on collateral compounds involved in the signal pathway. Further, the expression of genes that are up- or down-regulated in response to the enzyme protein signal pathway can also be assayed. In this embodiment the regulatory regions of these genes can be operably linked to a reporter gene such as luciferase.
Thus, modulators of enzyme gene expression can be identified in a method wherein a cell is contacted with a candidate compound and the expression of mRNA determined. The level of expression of enzyme mRNA in the presence of the candidate compound is compared to the level of expression of enzyme mRNA in the absence of the candidate compound. The candidate compound can then be identified as a modulator of nucleic acid expression based on this comparison and be used, for example to treat a disorder characterized by aberrant nucleic acid expression. When expression of mRNA is statistically significantly greater in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of nucleic acid expression. When nucleic acid expression is statistically significantly less in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of nucleic acid expression.
The invention further provides methods of treatment, with the nucleic acid as a target, using a compound identified through drug screening as a gene modulator to modulate enzyme nucleic acid expression in cells and tissues that express the enzyme. Modulation includes both up-regulation (i.e. activation or agonization) or down-regulation (suppression or antagonization) or nucleic acid expression.
Alternatively, a modulator for enzyme nucleic acid expression can be a small molecule or drug identified using the screening assays described herein as long as the drug or small molecule modulates the enzyme nucleic acid expression in the cells and tissues that express the protein.
The nucleic acid molecules are also useful for monitoring the effectiveness of modulating compounds on the expression or activity of the enzyme gene in clinical trials or in a treatment regimen. Thus, the gene expression pattern can serve as an indication of the continuing effectiveness of treatment with the compound, particularly with compounds to which a patient can develop resistance. The gene expression pattern can also indicate the physiological response of the affected cells to the compound. Accordingly, such monitoring would allow either increased administration of the compound or the administration of alternative compounds to which the patient has not become resistant. Similarly, if the level of nucleic acid expression falls below a desirable level, administration of the compound could be commensurately decreased.
The nucleic acid molecules are also useful in diagnostic assays for qualitative changes in enzyme nucleic acid expression, and particularly in qualitative . changes that lead to pathology. The nucleic acid molecules can be used to detect mutations in enzyme genes and gene expression products such as mRNA. The nucleic acid molecules can be used as hybridization probes to detect naturally occurring genetic mutations in the enzyme gene and thereby to determine whether a subject with the mutation is at risk for a disorder caused by the mutation. Mutations include deletion, addition, or substitution of one or more nucleotides in the gene, chromosomal rearrangement, such as inversion or transposition, modification of genomic DNA, such as aberrant methylation patterns or changes in gene copy number, such as amplification. Detection of a mutated form of the enzyme gene associated with a dysfunction provides a diagnostic tool for an active disease or susceptibility to disease when the disease results from overexpression, underexpression, or altered expression of an enzyme protein.
Individuals carrying mutations in the enzyme gene can be detected at the nucleic acid level by a variety of techniques. Genomic DNA can be analyzed directly or can be amplified by using PCR prior to analysis. RNA or cDNA can be used in the same way. In some uses, detection of the mutation may involve the use of such conventional methods as the use of a probe/primer in a polymerase chain reaction (PCR) such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (the latter of which can be particularly useful for detecting point mutations in the gene). This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a gene under conditions such that hybridization and amplification of the gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. Deletions and insertions can be detected by a change in size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to normal RNA or antisense DNA sequences. Alternatively, mutations in an enzyme gene can be directly identified, for example, by alterations in restriction enzyme digestion patterns determined by gel electrophoresis.
Further, sequence-specific ribozymes can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site. Perfectly matched sequences can be distinguished from mismatched sequences by nuclease cleavage digestion assays or by differences in melting temperature according to conventional methods.
Sequence changes at specific locations can also be assessed by nuclease protection assays such as RNase and S1 protection or the chemical cleavage method. Furthermore, sequence differences between a mutant enzyme gene and a wild-type gene can be determined by direct DNA sequencing. A variety of automated sequencing procedures can be utilized when performing the diagnostic assays, including, e.g., sequencing by mass spectrometry as disclosed in PCT International Publication No. WO 94/16101; Cohen et al., 1996, Adv. Chromatogr. 36: 127-162; and Griffin et al., 1993, Appl. Biochem. Biotechnol. 38: 147-159).
Other methods for detecting mutations in the gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA duplexes, electrophoretic mobility of mutant and wild- type nucleic acid is compared and movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis, techniques which are all familiar to one of skill in the art. Examples of other conventional methods for detecting point mutations include selective oligonucleotide hybridization, selective amplification, and selective primer extension. The nucleic acid molecules are also useful for testing an individual for a genotype that while not necessarily causing the disease, nevertheless affects the treatment modality. Thus, the nucleic acid molecules can be used to study the relationship between an individual's genotype and the individual's response to a compound used for treatment (pharmacogenomic relationship). Accordingly, the nucleic acid molecules described herein can be used to assess the mutation content of the enzyme gene in an individual in order to select an appropriate compound or dosage regimen for treatment.
Thus, nucleic acid molecules displaying genetic variations that affect treatment provide a diagnostic target that can be used to tailor treatment in an individual. Accordingly, the production of recombinant cells and animals containing these polymorphisms allow effective clinical design of treatment compounds and dosage regimens.
It is contemplated herein that a therapeutic effect may be achieved by controlling the expression of the nucleic acids of the present invention, for example, where a disorder is characterized by an abnormal or undesired RHAU nucleic acid expression. Thus, the present invention includes modulators of the nucleic acids encoding the RHAU proteins described herein. Such modulators include ribozymes, antisense oligonucleotides, triple helix DNA, RNA aptamers, siRNA and/or double stranded RNA directed to an appropriate nucleotide sequence encoding the RHAU proteins disclosed herein. These inhibitory molecules may be created using conventional techniques by one of skill in the art without undue burden or experimentation. For example, modifications (e.g. inhibition) of gene expression can be obtained by designing antisense molecules, DNA or RNA, to the control regions of the genes encoding the polypeptides discussed herein, i.e., to promoters, enhancers, and introns, which can act to prevent transcription and production of enzyme protein. For example, oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site may be used. Notwithstanding, all regions of the gene may be used to design an antisense molecule in order to create those which give strongest hybridization to the mRNA and such suitable antisense oligonucleotides may be produced and identified by standard assay procedures familiar to one of skill in the art.
Similarly, inhibition of the expression of gene expression may be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature (Gee JE et al., In: Huber BE and Carr Bl, eds, Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y., 1994). These molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to inhibit gene expression by catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples which may be used include engineered "hammerhead" or "hairpin" motif ribozyme molecules that can be designed to specifically and efficiently catalyze endonucleolytic cleavage of gene sequences, for example, a gene for RHAU.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
Ribozyme methods include exposing a cell to ribozymes or inducing expression in a cell of such small RNA ribozyme molecules (Grassi and Marini, 1996, Annals of Medicine 28: 499-510; Gibson, 1996, Cancer and Metastasis Reviews 15: 287-299). Intracellular expression of hammerhead and hairpin ribozymes targeted to mRNA corresponding to at least one of the genes discussed herein can be utilized to inhibit protein encoded by the gene.
Ribozymes can either be delivered directly to cells, in the form of RNA oligonucleotides incorporating ribozyme sequences, or introduced into the cell as an expression vector encoding the desired ribozymal RNA. Ribozymes can be routinely expressed in vivo in sufficient number to be catalytically effective in cleaving mRNA, and thereby modifying mRNA abundance in a cell (Cotten et al., 1989, EMBO J. 8: 3861-3866). In particular, a ribozyme coding DNA sequence, designed according to conventional, well known rules and synthesized, for example, by standard phosphoramidite chemistry, can be ligated into a restriction enzyme site in the anticodon stem and loop of a gene encoding a tRNA, which can then be transformed into and expressed in a cell of interest by methods routine in the art. Preferably, an inducible promoter (e.g., a glucocorticoid or a tetracycline response element) is also introduced into this construct so that ribozyme expression can be selectively controlled. For saturating use, a highly and constituently active promoter can be used. tDNA genes (i.e., genes encoding tRNAs) are useful in this application because of their small size, high rate of transcription, and ubiquitous expression in different kinds of tissues.
Therefore, ribozymes can be routinely designed' to cleave virtually any mRNA sequence, and a cell can be routinely transformed with DNA coding for such ribozyme sequences such that a controllable and catalytically effective amount of the ribozyme is expressed. Accordingly the abundance of virtually any RNA species in a cell can be modified or perturbed.
Ribozyme sequences can be modified in essentially the same manner as described for antisense nucleotides, e.g., the ribozyme sequence can comprise a modified base moiety.
RNA aptamers can also be introduced into or expressed in a cell to modify RNA abundance or activity. RNA aptamers are specific RNA ligands for proteins, such as for Tat and Rev RNA (Good et al., 1997, Gene Therapy 4: 45-54) that can specifically inhibit their translation.
Gene specific inhibition of gene expression may also be achieved using conventional RNA Interference and double stranded RNA technologies. A description of such technology may be found in, for example, Fire et al., 1998, Nature 391: 806-811 ; Agrawal et al., 2003, Microbiol. MoI. Biol. Rev. 67: 657-685 and WO 99/32619 which are hereby incorporated by reference in their entirety.
Antisense molecules, triple helix DNA, RNA aptamers, dsRNA, siRNA and ribozymes of the present invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the genes of the polypeptides discussed herein. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells, or tissues.
It is contemplated herein that the present invention includes the nucleic acid modulators discussed above as well as pharmaceutical compositions comprising these modulators. Similarly, it is contemplated herein that one can modulate the function and/or expression of a gene for a related regulatory protein or protein modified by RHAU as a way to produce a therapeutic effect by designing, for example, antibodies to these proteins and/or designing inhibitory antisense oligonucleotides, dsRNA, siRNA, triple helix DNA, ribozymes and RNA aptamers targeted to the genes for such proteins according to conventional methods. Pharmaceutical compositions comprising such modulators are also contemplated.
The invention also encompasses kits for detecting the presence of a RHAU nucleic acid in a biological sample. For example, the kit can comprise reagents such as a labeled nucleic acid or a nucleic acid that may be labeled or agent capable of detecting enzyme nucleic acid in a biological sample; means for determining the amount of enzyme nucleic acid in the sample; and means for comparing the amount of enzyme nucleic acid in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect enzyme protein mRNA or DNA.
Nucleic Acid Arrays
The present invention further provides nucleic acid detection kits, such as arrays or microarrays of nucleic acid molecules that are based on the sequence information provided herein in SEQ ID NO:1 and SEQ ID NO:2.
As used herein "arrays" or "microarrays" refer to an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon or other type of membrane, filter, chip, glass slide, or any other suitable solid support. In one embodiment, the microarray is prepared and used according to the methods described in U.S. Pat. No. 5,837,832 (Chee et al.), PCT application WO 95/11995 (Chee et al.), Lockhart DJ et al. (Nat. Biotech. 14: 1675-1680, 1996) and Schena M et al. (Proc. Natl. Acad. Sci. 93: 10614-10619, 1996), all of which are incorporated herein in their entirety by reference. In other embodiments, such arrays are produced by the methods described by Brown et al. (U.S. Pat. No. 5,807,522).
The microarray or detection kit is preferably composed of a large number of unique, single-stranded nucleic acid sequences, usually either synthetic antisense oligonucleotides or fragments of cDNAs, fixed to a solid support. The oligonucleotides are preferably about 6-60 nucleotides in length, more preferably 15-30 nucleotides in length, and most preferably about 20-25 nucleotides in length. For a certain type of microarray or detection kit, it may be preferable to use oligonucleotides that are only 7-20 nucleotides in length. The microarray or detection kit may contain oligonucleotides that cover the known 51, or 31, sequence, sequential oligonucleotides which cover the full length sequence; or unique oligonucleotides selected from particular areas along the length of the sequence. Polynucleotides used in the microarray or detection kit may be oligonucleotides that are specific to a gene or genes of interest.
In order to produce oligonucleotides to a known sequence for a microarray or detection kit, the gene(s) of interest is typically examined using a computer algorithm which starts at the 51 or at the 31 end of the nucleotide sequence. Typical algorithms will then identify oligomers of defined length that are unique to the gene, have a GC content within a range suitable for hybridization, and lack predicted secondary structure that may interfere with hybridization. In certain situations it may be appropriate to use pairs of oligonucleotides on a microarray or detection kit. The "pairs" will be identical, except for one nucleotide that preferably is located in the center of the sequence. The second oligonucleotide in the pair (mismatched by one) serves as a control. The number of oligonucleotide pairs may range from two to one million. The oligomers are synthesized at designated areas on a substrate using a light-directed chemical process. The substrate may be paper, nylon or other type of membrane, filter, chip, glass slide or any other suitable solid support. In another aspect, an oligonucleotide may be synthesized on the surface of the substrate by using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO 95/251116 (Baldeschweiler et al.) which is incorporated herein in its entirety by reference. In another aspect, a "gridded" array analogous to a dot (or slot) blot may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedures. An array, such as those described above, may be produced by hand or by using available devices (slot blot or dot blot apparatus), materials (any suitable solid support), and machines (including robotic instruments), and may contain 8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other number between two and one million which lends itself to the efficient use of commercially available instrumentation.
In order to conduct sample analysis using a microarray or detection kit, the RNA or DNA from a biological sample is made into hybridization probes. The mRNA is isolated, and cDNA is produced and used as a template to make antisense RNA. The antisense RNA is amplified in the presence of fluorescent nucleotides, and labeled probes are incubated with the microarray or detection kit so that the probe sequences hybridize to complementary oligonucleotides of the microarray or detection kit. Incubation conditions are adjusted so that hybridization occurs with precise complementary matches or with various degrees of less complementarity. After removal of non-hybridized probes, a scanner is used to determine the levels and patterns of fluorescence. The scanned images are examined to determine degree of complementarity and the relative abundance of each oligonucleotide sequence on the microarray or detection kit. The biological samples may be obtained from any bodily fluid, cultured cells, biopsies, or other tissue preparations. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously. This data may be used for large-scale correlation studies on the sequences, expression patterns, mutations, variants, or polymorphisms among samples.
Using such arrays, the present invention provides methods to identify the expression of the enzyme proteins/peptides of the present invention. In detail, such methods comprise incubating a test sample with one or more nucleic acid molecules and assaying for binding of the nucleic acid molecule with components within the test sample. Such assays will typically involve arrays comprising many genes, at least one of which is a gene of the present invention and or alleles of the enzyme gene of the present invention.
Conditions for incubating a nucleic acid molecule with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the nucleic acid molecule used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or array assay formats can readily be adapted to employ the nucleic acid sequences disclosed herein. Examples of such assays can be found in Chard T, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands, 1986, Bullock G R et al., Techniques in Immunocytochemistry, Academic Press, Orlando, FIa., Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985), Tijssen P, Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands, 1985.
The test samples of the present invention include cells, protein or membrane extracts of cells. The test sample used in the above-described method will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing nucleic acid extracts or of cells are well known in the art and can be readily adapted in order to obtain a sample that is compatible with the system utilized. In another embodiment of the present invention, kits are provided which contain the necessary reagents to carry out the assays of the present invention.
Specifically, the invention provides a compartmentalized kit to receive, in close confinement, one or more containers which comprise: (a) a first container comprising one of the nucleic acid molecules that can bind to a fragment of the human genome disclosed herein; and (b) one or more other containers comprising one or more of the following: wash reagents, reagents capable of detecting presence of a bound nucleic acid.
In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers, strips of plastic, glass or paper, or arraying material such as silica. Such containers allow one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the nucleic acid probe, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound probe. One skilled in the art will readily recognize that the previously unidentified enzyme gene of the present invention can be routinely identified using the sequence information disclosed herein and can be readily incorporated into one of the established kit formats which are well known in the art, particularly expression arrays.
Vectors/Host Cells
The invention also provides vectors containing the nucleic acid molecules described herein. The term "vector" refers to a vehicle, preferably a nucleic acid molecule, which can transport the nucleic acid molecules. When the vector is a nucleic acid molecule, the nucleic acid molecules are covalently linked to the vector nucleic acid. With this aspect of the invention, the vector includes a plasmid, single or double stranded phage, a single or double stranded RNA or DNA viral vector, or artificial chromosome, such as a BAC, PAC, YAC, or MAC.
A vector can be maintained in the host cell as an extrachromosomal element where it replicates and produces additional copies of the nucleic acid molecules. Alternatively, the vector may integrate into the host cell genome and produce additional copies of the nucleic acid molecules when the host cell replicates.
The invention provides vectors for the maintenance (cloning vectors) or vectors for expression (expression vectors) of the nucleic acid molecules. The vectors can function in prokaryotic or eukaryotic cells or in both (shuttle vectors).
Expression vectors contain cis-acting regulatory regions that are operably linked in the vector to the nucleic acid molecules such that transcription of the nucleic acid molecules is allowed in a host cell. The nucleic acid molecules can be introduced into the host cell with a separate nucleic acid molecule capable of affecting transcription. Thus, the second nucleic acid molecule may provide a trans-acting factor interacting with the cis-regulatory control region to allow transcription of the nucleic acid molecules from the vector. Alternatively, a transacting factor may be supplied by the host cell. Finally, a trans-acting factor can be produced from the vector itself. It is understood, however, that in some embodiments, transcription and/or translation of the nucleic acid molecules can occur in a cell-free system.
The regulatory sequence to which the nucleic acid molecules described herein can be operably linked include promoters for directing mRNA transcription. These include, but are not limited to, the left promoter from bacteriophage λ, the lac, TRP, and TAC promoters from E. coli, the early and late promoters from SV40, the CMV immediate early promoter, the adenovirus early and late promoters, and retrovirus long-terminal repeats.
In addition to control regions that promote transcription, expression vectors may also include regions that modulate transcription, such as repressor binding sites and enhancers. Examples include the SV40 enhancer, the cytomegalovirus immediate early enhancer, polyoma enhancer, adenovirus enhancers, and retrovirus LTR enhancers.
In addition to containing sites for transcription initiation and control, expression vectors can also contain sequences necessary for transcription termination and, in the transcribed region, a ribosome binding site for translation. Other regulatory control elements for expression include initiation and termination codons as well as polyadenylation signals. One of ordinary skill in the art would be aware of the numerous regulatory sequences that are useful in expression vectors. Such regulatory sequences are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
A variety of expression vectors can be used to express a nucleic acid molecule. Such vectors include chromosomal, episomal, and virus-derived vectors, for example vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, including yeast artificial chromosomes, from viruses such as baculoviruses, papovaviruses such as SV40, Vaccinia viruses, adenoviruses, poxviruses, pseudorabies viruses, and retroviruses. Vectors may also be derived from combinations of these sources such as those derived from plasmid and bacteriophage genetic elements, e.g. cosmids and phagemids. Appropriate cloning and expression vectors for prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The regulatory sequence may provide constitutive expression in one or more host cells (i.e. tissue specific) or may provide for inducible expression in one or more cell types such as by temperature, nutrient additive, or exogenous factor such as a hormone or other ligand. A variety of vectors providing for constitutive and inducible expression in prokaryotic and eukaryotic hosts are well known to those of ordinary skill in the art.
The nucleic acid molecules can be inserted into the vector nucleic acid by well- known methodology. Generally, the DNA sequence that will ultimately be expressed is joined to an expression vector by cleaving the DNA sequence and the expression vector with one or more restriction enzymes and then ligating the fragments together. Procedures for restriction enzyme digestion and ligation are well known to those of ordinary skill in the art.
The vector containing the appropriate nucleic acid molecule can be introduced into an appropriate host cell for propagation or expression using well-known techniques. Bacterial cells include, but are not limited to, E. coli, Streptomyces, and Salmonella typhimurium. Eukaryotic cells include, but are not limited to, yeast, insect cells such as Drosophila, animal cells such as COS and CHO cells, and plant cells.
As described herein, it may be desirable to express the peptide as a fusion protein. Accordingly, the invention provides fusion vectors that allow for the production of the peptides. Fusion vectors can increase the expression of a recombinant protein, increase the solubility of the recombinant protein, and aid in the purification of the protein by acting for example as a ligand for affinity purification. A proteolytic cleavage site may be introduced at the junction of the fusion moiety so that the desired peptide can ultimately be separated from the fusion moiety. Proteolytic enzymes include, but are not limited to, factor Xa, thrombin, and enteroenzyme. Typical fusion expression vectors are well known in the art and include pGEX, pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc and pET 11d.
Recombinant protein expression can be maximized in host bacteria according to conventional methods, e.g., by providing a genetic background wherein the host cell has an impaired capacity to proteolytically cleave the recombinant protein. Alternatively, the sequence of the nucleic acid molecule of interest can be altered to provide preferential codon usage for a specific host cell, for example E. coli.
The nucleic acid molecules can also be expressed by expression vectors that are operative in yeast. Examples of vectors for expression in yeast, e.g., S. cerevisiae, are familiar to one of skill in the art and include pYepSed , pMFa, pJRY88 and pYES2 (Invitrogen Corporation, San Diego, Calif.).
The nucleic acid molecules can also be expressed in insect cells using, for example, baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) are familiar to one of skill in the art and include the pAc series and the pVL series.
In certain embodiments of the invention, the nucleic acid molecules described herein are expressed in mammalian cells using mammalian expression vectors. Examples of mammalian expression vectors and methods of their use are also familiar to one of skill in the art and include pCDM8 and pMT2PC.
The expression vectors listed herein are provided by way of example only and a person of ordinary skill in the art would be aware of other vectors suitable for maintenance propagation or expression of the nucleic acid molecules described herein. The invention also encompasses vectors in which the nucleic acid sequences described herein are cloned into the vector in reverse orientation, but operably linked to a regulatory sequence that permits transcription of antisense RNA. Thus, an antisense transcript can be produced to all, or to a portion, of the nucleic acid molecule sequences described herein, including both coding and non-coding regions. Expression of this antisense RNA is subject to each of the parameters described above in relation to expression of the sense RNA (regulatory sequences, constitutive or inducible expression, tissue-specific expression).
The invention also relates to recombinant host cells containing the vectors described herein. Host cells therefore include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as mammalian cells.
The recombinant host cells are prepared by introducing the vector constructs described herein into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Host cells can contain more than one vector. Thus, different nucleotide sequences can be introduced on different vectors of the same cell. Similarly, the nucleic acid molecules can be introduced either alone or with other nucleic acid molecules that are not related to the nucleic acid molecules such as those providing trans-acting factors for expression vectors. When more than one vector is introduced into a cell, the vectors can be introduced independently, co- introduced or joined to the nucleic acid molecule vector.
In the case of bacteriophage and viral vectors, these can be introduced into cells as packaged or encapsulated virus by standard procedures for infection and transduction. Viral vectors can be replication-competent or replication-defective. In the case in which viral replication is defective, replication will occur in host cells providing functions that complement the defects.
Vectors generally include selectable markers that enable the selection of the subpopulation of cells that contain the recombinant vector constructs. The marker can be contained in the same vector that contains the nucleic acid molecules described herein or may be on a separate vector. Markers include tetracycline or ampicillin-resistance genes for prokaryotic host cells and dihydrofolate reductase or neomycin resistance for eukaryotic host cells. However, any marker that provides selection for a phenotypic trait will be effective.
While the mature proteins can be produced in bacteria, yeast, mammalian cells, and other cells under the control of the appropriate regulatory sequences, cell- free transcription and translation systems can also be used to produce these proteins using RNA derived from the DNA constructs described herein. Where secretion of the peptide is desired but difficult to achieve with multi- transmembrane domain containing proteins such as enzymes, appropriate secretion signals are incorporated into the vector. The signal sequence can be endogenous to the peptides or heterologous to these peptides.
Where the peptide is not secreted into the medium, which is typically the case with enzymes, the protein can be isolated from the host-cell by standard disruption procedures, including freeze thaw, sonication, mechanical disruption, use of lysing agents and the like. The peptide can then be recovered and purified by well-known purification methods. It is also understood that depending upon the host cell utilized for recombinant production of the peptides described herein, the peptides can have various glycosylation patterns, depending upon the cell, or may be non-glycosylated as when produced in bacteria. In addition, the peptides may include an initial modified methionine in some cases as a result of a host-mediated process.
Uses of Vectors and Host Cells
Recombinant host cells expressing the peptides described herein have a variety of uses. For example, the cells may be used for producing an enzyme protein or peptide that can be further purified to obtain desired amounts of enzyme protein or fragments. Host cells are also useful for conducting cell-based assays involving enzyme protein or enzyme protein fragments, such as those described above as well as other formats known in the art. Thus, a recombinant host cell expressing a native enzyme protein is useful for assaying compounds that stimulate or inhibit enzyme protein function.
Host cells are also useful for identifying enzyme protein mutants including those with impaired function. If the mutants naturally occur and give rise to a pathology, host cells containing the mutations may be used to assay compounds that have a desired effect on the mutant enzyme protein (for example, stimulating or inhibiting function) which may not be indicated by their effect on the native enzyme protein.
Genetically engineered host cells can be further used to produce non-human transgenic animals. A transgenic animal is preferably a mammal, for example a rodent, such as a rat or mouse, in which one or more of the cells of the animal include a transgene. These animals are useful for studying the function of an enzyme protein and identifying and evaluating modulators of enzyme protein activity. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians. Any of the enzyme protein nucleotide sequences can be introduced as a transgene into the genome of a non-human animal, e.g., a mouse and any of the regulatory or other sequences useful in expression vectors can form part of the transgenic sequence. This includes intronic sequences and polyadenylation signals, if not already included. A tissue-specific regulatory sequence(s) can be operably linked to the transgene to direct expression of the enzyme protein to particular cells.
Various methods for generating transgenic animals have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. These methods also include the cre/loxP recombinase system of bacteriophage P1 (Lakso et al., 1992, PNAS 89: 6232-6236) and the FLP recombinase system of S. cerevisiae (O'Gorman et al., 1991 , Science 251 : 1351-1355).
Clones of transgenic animals can also be produced according to the methods described in Wilmut I. et al. (Nature 385: 810-813, 1997) and PCT International Publication Nos. WO 97/07668 and WO 97/0766.
Transgenic animals containing recombinant cells that express the peptides described herein are useful to assay in vivo enzyme protein function, including substrate interaction, the effect of specific mutant enzyme proteins on enzyme protein function and substrate interaction, and the effect of chimeric enzyme proteins. It is also possible to use the transgenic animals described herein to assess the effect of null mutations, that is, mutations that substantially or completely eliminate one or more enzyme protein functions.
Pharmaceutical Compositions
As discussed above, the present invention includes pharmaceutical compositions comprising, e.g., modulators of RHAU enzyme protein activity or nucleic acid expression which may be used to treat a subject with a RHAU related disorder, e.g., neoplasia. The pharmaceutical compositions disclosed herein are to be administered to a patient at therapeutically effective doses to treat or ameliorate such disorders. A therapeutically effective dose refers to that amount of the compound sufficient to result in amelioration of said disorder.
The pharmaceutical compositions for use in accordance with the present invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.
Thus, modulators and/or compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or topical, oral, buccal, parenteral or rectal administration.
For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl- p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
For buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example, antisense oligonucleotides, triple helix DNA, ribozymes, RNA aptamer and double stranded RNA designed to inhibit RHAU gene expression, antibodies to RHAU or related regulatory proteins or fragments thereof, useful to treat and/or ameliorate the pathological effects of a RHAU related disorder. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors that may be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. Pharmaceutical formulations suitable for oral administration of proteins are described, e.g., in U.S. Patents 5,008,114; 5,505,962; 5,641 ,515; 5,681 ,811 ; 5,700,486; 5,766,633; 5,792,451 ; 5,853,748; 5,972,387; 5,976,569; and 6,051 ,561.
It is contemplated that the invention described herein is not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention in any way.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to the "antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth. Unless otherwise apparent from context, the term "RHAU" as used herein is meant to include RHAUΔ14and any other isoforms of RHAU.
Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are described. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing the materials and methodologies that are reported in the publication which might be used in connection with the invention.
In practicing the present invention, many conventional techniques in molecular biology are used. These techniques are well known and are explained in, for example, Current Protocols in Molecular Biology, Volumes I, II, and III, 1997 (Ausubel, FM, ed.); Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989 (Sambrook et al.); DNA Cloning: A Practical Approach, Volumes I and II, 1985 (Glover, DN, ed.); Oligonucleotide Synthesis, 1984 (Gait, ML, ed.); Nucleic Acid Hybridization, 1985, (Hames and Higgins, eds); Transcription and Translation, 1984 (Hames and Higgins, eds); Animal Cell Culture, 1986 (Freshney, Rl, ed.); Immobilized Cells and Enzymes, IRL Press, 1986; Perbal, 1984, A Practical Guide to Molecular Cloning; the series, Methods in Enzymology, Academic Press, Inc.; Gene Transfer Vectors for Mammalian Cells, 1987 (Miller, JH and Calos, MP, eds, Cold Spring Harbor Laboratory); and Methods in Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds, respectively).
The following examples further illustrate the present invention and are not intended to limit the invention.
EXAMPLES
EXAMPLE 1
Identification, Isolation and Functional Characterization of RHAU and
RHAUΔ14
RHAU was isolated in association with the AU-rich element (ARE) of urokinase plasminogen activator mRNA (AREuPA) and further characterized as described below.
Experimental Methods
Plasmids
The protein coding regions of RHAU and RHAUΔ14cDNAs are amplified by RT-
PCR using total RNA isolated from THP- 1 cells and inserted between the EcoR1 and Xho1 sites of pc DNA3-HA (Kisielow et al., 2002, Biochem. J. 363: 1-5).
RHAU and RHAUΔ14 E335A mutants are created using QuickChange (Stratagene, La JoIIa, CA) and suitable oligonucleotide primers. To express GST- RHAU fusion proteins, the protein coding regions of various RHAU and RHAUΔU cDNAs are subcloned between the EcoR1 and Xho1 sites of the baculovirus expression vector pAcGHLT-A (Pharmingen, San Diego, CA). Expression vectors for GST, GST-HuR, and tetracycline-regulated β-globin or chimeric β- globin-AREuPA mRNAs are described in Tran et al., 2003, MoI. Cell. Biol. 23: 7177-7188.
Cell Treatments
Cells are maintained and transiently or stably transfected as described in Tran et al., 2003, MoI. Cell. Biol. 23: 7177-7188. Small interfering RNAs (siRNAs; Xeragon, Germantown, MD) used to target RHAU mRNA are as follows: sense, GGG AAC UGC GAA GAA GGU AUU-3' (SEQ ID NO:5) and antisense, UAC CUU CUU CGC AGU UCC CUU-31 (SEQ ID NO:6). These sequences contain 3' UU overhangs and target both RHAU isoforms. Control siRNA sequences: sense, GUA CCU GAC UAG UCG CAG AAG (SEQ ID NO:7); antisense, UCU GCG ACU AGU CAG GUA CGG (SEQ ID NO:8). Gene specificity is ensured by searching the oligoribonucleotide sequences using BLAST. Transfection of siRNA duplexes is performed as described in Kisielow et al., 2002, Biochem. J. 363: 1-5. Analysis in HeLa-Tet-Off cells of chimeric β-globin and endogenous uPA/uPAR mRNA stability, total RNA isolation, Northern blotting, and hybridization with specific,32P-labeled cDNA probes are performed as described in Tran et al., 2003, MoI. Cell. Biol. 23: 7177-7188. For immunofluorescence microscopy, cells are processed and incubated with anti-HA monoclonal antibodies (12CA5) as described in Tran et al., 2003, MoI. Cell. Biol. 23: 7177- 7188.
In Vitro Synthesis of RNA Transcripts
AREuPA wild-type or mutant sequences are prepared from annealed DNA oligonucleotides containing T7 promoter sequences as described in Tran et al., 2003, MoI. Cell. Biol. 23: 7177-7188. A control RNA sequence derived from the SV40 large T antigen is similarly prepared (only sense strand is shown): TAA TAC GAC TCA CTA TAG GGT GCA ATG TAC TTG CAA AGA ATG GCC TGA GTG TGC AAA GAA AAT GTC TGC T (SEQ ID NO:9). For cell -free RNA decay assays, capped and polyadenylated (A60),32P-labeled RNA transcripts composed of 140 nucleotides of the rabbit β-globin 3' UTR without or with the 47 nucleotides AREuPA are prepared as detailed previously (Fritz et al., 2000, Sci. STKE 2000, PU ).
AR E- Affinity Chromatography and Mass Spectrometry
Non-labeled T7 transcripts are covalently linked to adipic acid dihydrazide agarose beads (Sigma) as described in Caputi et al., 1999, EMBO J. 18: 4060- 4067. RNA-bound beads are equilibrated in buffer D (Dignam et al., 1983, Nucleic Acids Res. 11: 1475-1489) and mixed with 1.5 mg of HeLa nuclear extract (Dignam et al., 1983, Nucleic Acids Res. 11 : 1475-1489) at 49C for 2 hrs. Unbound proteins are removed by washing six times with buffer D. Beads are boiled in SDS-sample buffer and eluted proteins analyzed by SDS-PAGE and Coomassie staining. Bands of interest are excised from the gel, reduced with DDT, alkylated with iodoacetaminde and cleaved with trypsin. The extracted tryptic peptides are desalted with 5% formic acid, 5% methanol in water on a 200 nl Poros P20 column and concentrated to 1 μl with 5% formic acid, 50% methanol in water directly into the Nanoelectrospray ionization (NanoESI) needle for NanoESI tandem mass spectrometry (WiIm and Mann, 1996, Anal. Chem. 68: 1-8). The mass spectra are obtained on an API 300 mass spectrometer (PE Sciex, Concord, ON, Canada) equipped with a NanoESI source (Protana, Odense, Denmark). Proteins are identified as described in Mann and WiIm, 1994, Anal. Chem. 66: 4390-4399.
Recombinant Protein Expression
Glutathione S-transferase (GST) and GST-HuR proteins are expressed in E. coli BL21(DE3) and purified using glutathione-Sepharose (Amersham, Little Chalfont, UK) according to the supplier's instructions. GST-RHAU proteins are expressed in Sf9 cells according to the supplier's instructions (Pharmingen) and purified as above.
Cell-Free RNA Decay
HeLa S100 extracts are prepared as described in Fritz et al., 2000, Sci. STKE
2000, PL1 , with the exception that adherent HeLa cells are used. A premix containing 50 μg of total HeLa S100 proteins in IVDA buffer (Wang and Kiledjian,
2001 , Cell 107: 751-762) and, where indicated, 100 ng of various recombinant GST fusion proteins is prepared (45μl for five reactions) on ice. Nine μl aliquots of the premix are incubated at 37°C with 1 μl (103 cpm) of32P-RNA substrates. Reactions are stopped at different times with the addition of 200 μl of a high-salt buffer (Fritz et al., 2000, Sci. STKE 2000, PL1), 10μg of calf liver tRNA, and32 P- AREuPA RNA (103cpm), used as an internal control for the subsequent phenol/chloroform extraction and ethanol precipitation of the processed RNA. Precipitated RNA is resuspended in loading buffer, resolved on a 7 M urea/5% acrylamide gel and analyzed with a Phosphorimager (Molecular Dynamics, Sunnyvale, CA).
lmmunoprecipation and GST Pull-Down Assays lmmunoprecipitations are performed using HeLa whole-cell extracts (500 μg), in the absence of presence of RNases A (10 μg/ml) and T1 (100 U/ml), together with 4 μg of a mouse anti-HA monoclonal antibody (12CA5) or 10 μl of anti- hRrp40p, anti-PM-Scl100, and anti-PARN rabbit anti-sera precoupled to a 50% solution of protein A-Sepharose beads (100 μl; Amersham) for 2 hour at 4° C. GST pull-down assays are performed with 10 μg of GST or GST-HuR coupled to a 50% solution of glutathione-Sepharose 4B beads (100 μl; Amersham) and incubated with HeLa extracts as described above. Precipitated proteins are analyzed by SDS-PAGE and Western blotting. In addition to the antibodies described above, affinity-purified rabbit polyclonal antibody raised against a synthetic peptide corresponding to 17 amino acids (RHAU, 991-1007) at the C terminus of RHAU (Davids Biotechnologie, Regensburg, Germany) and the monoclonal antibodies against PKR (p68 kinase; Transduction Laboratories) and DRBP76 (NFAR; BD Biosciences, Bedford, MA) for Westerns are used.
ATPase Assay
Assays are performed as described (Kang et al., 2002, PNAS USA 99: 637-642) with modifications. Each 20 μl reaction contains 50 mM MOPS, pH 6.5, 3 mM MgCI2, 2mM DTT, 0.1 mM ATP1 5 μCi of [γ-32P]ATP (3,000 Ci/mmol; Amersham), 200 ng GST, or GST-RHAU, in the absence or presence of the indicated DNA or RNA species (20 ng/μl). Reactions are incubated at 37°C for 30 min., spotted on polyethylene-immine (PEI) cellulose (Merck, Whitehouse Station, NJ) and developed in 0.75 M LiCI/1 M formic acid solution for 20 min. Visualization and quantitation of radioactive signals are performed with a phosphorimager.
Results
Identification of Proteins Specifically Interacting with the AREuPA
RNAs corresponding to the ARE sequence of uPA mRNA (ARE WT), a mutated ARE sequence (ARE MUT), or a control SV40 early message sequence (C RNA) are coupled to agarose beads and used to affinity purify factors from HeLa nuclear extracts. Nuclear proteins which bound to the immobilized RNAs are analyzed by SDS-PAGE. Three proteins are enriched in the ARE WT fraction compared to the ARE MUT or control RNA fraction, or beads alone. These proteins are excised from the gel and further characterized by NanoESI tandem mass spectrometry. Comparison of tryptic peptides against a peptide database (Mann and WiIIm, 1994, Anal. Chem. 66: 4390-4399) identified the proteins as HuR, NFAR1 and MLE-like 1 (MLEL1) which we renamed RHAU (see below). MLEL1 (accession number AF217190) was named because of sequence similarity to Drosophila MLE (maleless) - a gene required for dosage compensation (Kuroda et al., 1991 , Cell 66: 935-947). However, our homology searches and alignments of predicted amino acid full-length sequences from different organisms reveal that in Drosophila, there is an uncharacterized sequence (accession number AAF53921) more similar to MLEL1 than MLE. Nevertheless, the biological function of MLEL1 , much less whether it is functionally related to MLE, had not previously been addressed. To reflect its biochemical nature that the protein was isolated from ARE binding fractions, we refer to the protein as RHAU (RNA Λelicase associated with AU-ήch element).
RHAU is a DExH/D RNA Helicase that is Expressed in Two isoforms
RHAU belongs to the DExH/D family of ATP-dependent RNA helicases and contains an evolutionary-conserved RNA helicase core region of approximately 440 amino acids which is composed of at least six discrete functional domains (data not shown). For functional studies, the protein coding region of RHAU is amplified by RT-PCR using total RNA isolated from the human monocytic leukemia cell line THP- 1. PCR products are cloned into a HA-tagged eukaryotic expression vector (pcDNA3-HA) and sequenced. Two isoforms of RHAU are obtained, which was confirmed by two independent RT-PCR experiments. Sequencing revealed that the longer isoform is identical to an entry in the database (MLEL1), while the shorter isoform lacked a stretch of 14 amino acids within the core region (amino acids 202-638) (data not shown). This deletion resulted from usage of an alternative splicing acceptor site 42 nucleotides downstream of the 5' end of exon 13 (data not shown). We referred to the shorter isoform as RHAUΔU. The amino acid and nucleic acid sequence information for RHAU and RHAUA14 is provided below:
Table 1 : RHAU Amino Acid Sequence (AJ577133; SEQ ID NO:3)
1 msydyhqnwg rdggprssgg gygggpaggh ggnrgsgggg ggggggrggr grhpghlkgr 61 eigmwyakkq ggknkeaerq erawhmder reegivqlln svgakndkes eaqiswfape 121 dhgygtevst kntpcsenkl diqekklinq ekkmfrirnr syidrdseyl Iqenepdgtl 181 dqklledlqk kkndlryiem qhfreklpsy gmgkelvnli dnhqvtvisg etgcgkttqv 241 tqfildnyie rgkgsacriv ctqprrisai svaervaaer aescgsgnst gyqirlqsrl 301 prkqgsilyc ttgiilqwlq sdpylssvsh ivldeihern lqsdvlmtw kdllnfrsdl 361 kvilmsatln aekfseyfgn cpmihipgft fpweylled viekiryvpe qkehrcqfkr
421 gfmqghvnrq ekeekeaiyk erwpdyvrel rrrysastvd viemmeddkv dlnlivalir
481 yivleeedga ilvflpgwdn istlhdllms qvmfksdkfl iiplhslmpt vnqtqvfkrt
541 ppgvrkivia tniaetsiti ddwyvidgg kikethfdtq nnistmsaew vskanakqrk
601 gragrvqpgh cyhlynglra sllddyqlpe ilrtpleelc Iqikilrlgg iayflsrlmd
661 ppsneavlls irhlmelnal dkqeeltplg vhlarlpvep higkmilfga lfccldpvlt
721 iaaslsfkdp fviplgkeki adarrkelak dtrsdhltw nafegweear rrgfryekdy
781 cweyflssnt lqmlhnmkgq faehllgagf vssrnpkdpe sninsdneki ikavicagly
841 pkvakirlnl gkkrkmvkvy tktdglvavh pksvnveqtd fhynwliyhl kmrtssiyly
901 dctevspycl Iffggdisiq kdndqetiav dewivfqspa riahlvkelr keldillqek
961 iesphpvdwn dtksrdcavl saiidliktq ekatprnfpp rfqdgyys
Amino acids in bold indicate amino acids 517-530 missing in RHAU
Table 2: RHAUΔU Amino Acid Sequence (AJ577134; SEQ ID NO:4)
1 msydyhqnwg rdggprssgg gygggpaggh ggnrgsgggg ggggggrggr grhpghlkgr
61 esigmwyakkq gqknkeaerq erawhmder reeqivqlln svqakndkes eaqiswfape
121 dhgygtevst kntpcsenkl diqekklinq ekkmfrirnr syidrdseyl lqenepdgtl
181 dqklledlqk kkndlryiem qhfreklpsy gmqkelvnli dnhqvtvisg etgcgkttqv
241 tqfildnyie rgkgsacriv ctqprrisai svaervaaer aescgsgnst gyqirlqsrl
301 prkqgsilyc ttgiilqwlq sdpylssvsh ivldeihern lqsdvlmtw kdllnfrsdl
361 kvilmsatln aekfseyfgn cpmihipgft fpweylled viekiryvpe qkehrcqfkr
421 gfmqghvnrq ekeekeaiyk erwpdyvrel rrrysastvd viemmeddkv dlnlivalir
481 yivleeedga ilvflpgwdn istlhdllms qvmfksvnqt qvfkrtppgv rkiviatnia
541 etsitiddw yvidggkike thfdtqnnis tmsaewvska nakqrkgrag rvqpghcyhl
601 ynglraslld dyqlpeilrt pleelclqik ilrlggiayf lsrlmdppsn eavllsirhl
661 melnaldkqe eltplgvhla rlpvephigk milfgalfcc ldpvltiaas Isfkdpfvip
721 lgkekiadar rkelakdtrs dhltwnafe gweearrrgf ryekdycwey flssntlqml
781 hnmkgqfaeh llgagfvssr npkdpesnin sdnekiikav icaglypkva kirlnlgkkr
841 kmvkvytktd glvavhpksv nveqtdfhyn wliyhlkmrt ssiylydcte vspycllffg
901 gdisiqkdnd qetiavdewi vfqspariah Ivkelrkeld illqekiesp hpvdwndtks
961 rdcavlsaii dliktqekat prnfpprfqd gyys Table 3: RHAU Nucleic Acid Sequence (AJ577133: SEQ ID NO:1)
1 atgagttatg actaccatca gaactggggc cgtgatgggg gtccccgcag ctccggtggg
61 ggctatggag gggggccagc agggggtcat ggaggtaacc gaggctccgg aggaggcggc
121 ggcggcggag ggggtggtcg aggcggcagg ggccggcatc ccgggcacct gaaaggccgc
181 gaaatcggca tgtggtacgc gaaaaaacag gggcagaaga acaaggaagc ggagaggcaa
241 gagagagctg tagtacacat ggatgaacga cgagaagaac aaattgtaca gttactgaat
301 tctgttcaag cgaagaatga taaagagtca gaagcacaga tatcctggtt tgctcctgag
361 gatcatggat acggtactga agtttctact aagaacacac catgctcaga gaacaaactt
421 gacatccagg aaaagaagtt gataaatcaa gaaaaaaaaa tgtttagaat caggaacaga
481 tcatatattg accgagattc tgagtatctc ttgcaagaaa atgaaccaga tggaacttta
541 gaccaaaaat tattggaaga tttacaaaag aaaaaaaatg accttcggta tattgaaatg
601 cagcatttca gagaaaagct gccttcgtat ggaatgcaaa aggaattggt aaatttaatt
661 gataaccatc aggtaacagt aataagtggt gaaactggtt gtggcaaaac cactcaagtt
721 actcagttca ttttggataa ctacattgaa agaggaaaag gatctgcttg cagaatagtt
781 tgtactcagc caagaagaat tagtgccatt tcagttgcgg aaagagtagc tgcagaaagg
841 gcagaatctt gtggcagtgg taatagtact ggatatcaaa ttcgtctcca gagtcggttg
901 ccaaggaaac agggttctat cttatactgt acaacaggaa tcatccttca gtggctccag
961 tcagacccgt atttgtccag tgttagtcat atcgtacttg atgaaatcca tgaaagaaat
1021 ctgcagtcag atgttttaat gactgttgtt aaagaccttc tcaattttcg atctgacttg
1081 aaagtaatat tgatgagtgc aacattgaat gcagaaaagt tttcagaata ttttggtaac
1141 tgtccaatga tacatatacc tggttttacc tttccggttg tggaatatct tttggaagat
1201 gtaattgaaa aaataaggta tgttccagaa caaaaagaac acagatgcca gtttaagagg
1261 ggtttcatgc aagggcatgt aaatagacaa gaaaaagaag aaaaagaagc aatatataaa
1321 gaacgttggc cagattatgt aagggaactg cgaagaaggt attctgcaag tactgtagat
1381 gttatagaaa tgatggagga tgataaagtt gatctgaatt tgattgttgc cctcatccga
1441 tacattgttt tggaagaaga ggatggtgcg atactggtct ttctgccagg ctgggacaat
1501 atcagcactt tacatgatct cttgatgtca caagtaatgt ttaaatcaga taaattttta
1561 attatacctt tacattcact gatgcctaca gttaaccaga cacaggtgtt taaaagaacc
1621 cctcctggtg ttcggaaaat agtaattgct accaacattg cggagactag cattaccata
1681 gatgatgtcg tttatgtgat agatggagga aaaataaaag agacgcattt tgatactcag
1741 aacaatatca gtacaatgtc cgctgagtgg gttagtaaag ctaatgccaa acagagaaaa 1801 ggtcgagctg gaagagttca acctggtcat tgctatcatc tgtataatgg tcttagagca
1861 agtcttctag atgactatca actgccagaa attttgagaa ctcctttgga agaactttgt
1921 ttacaaataa agattttaag gctaggtgga attgcttatt ttctgagtag attaatggac
1981 ccaccatcaa atgaggcagt gttactctcc ataagacacc tgatggagct gaacgctttg
2041 gataaacaag aagaattgac acctcttgga gtccacttgg cacgattacc cgttgagcca
2101 catattggaa aaatgattct ttttggagca ctgttctgct gcttagaccc agtactcact
2161 attgctgcta gtctcagttt caaagatcca tttgtcattc cactgggaaa agaaaagatt
2221 gcagatgcaa gaagaaagga attggcaaag gatactagaa gtgatcactt aacagttgtg
2281 aatgcgtttg agggctggga agaggctagg cgacgtggtt tcagatacga aaaggactat
2341 tgctgggaat attttctgtc ttcaaacaca ctgcagatgc tgcataacat gaaaggacag
2401 tttgctgagc atcttcttgg agctggattt gtaagcagta gaaatcctaa agatccagaa
2461 tctaatataa attcagataa tgagaagata attaaagctg tcatctgtgc tggtttatat
2521 cccaaagttg ctaaaattcg actaaatttg ggtaaaaaaa gaaaaatggt aaaagtttac
2581 acaaaaaccg atggcctggt tgctgttcat cctaaatctg ttaatgtgga gcaaacagac
2641 tttcactaca actggcttat ctatcaccta aagatgagaa caagcagtat atacttgtat
2701 gactgcacag aggtttcccc atactgtctc ttgttttttg gaggtgacat ttccatccag
2761 aaggataacg atcaggaaac tattgctgta gatgagtgga ttgtatttca gtctccagca
2821 agaattgccc atcttgttaa ggaattaaga aaggaactag atattcttct gcaagagaag
2881 attgaaagtc ctcatcctgt agactggaat gacactaaat ccagagactg tgcagtactg
2941 tcagctatta tagacttgat caaaacacag gaaaaggcaa ctcccaggaa ctttccgcca
3001 cgattccagg atggatatta cagctga
Table 4: RHAUΔ14 Nucleic Acid Sequence (AJ577134; SEQ ID NO:2)
1 atgagttatg actaccatca gaactggggc cgtgatgggg gtccccgcag ctccggtggg
61 ggctatggag gggggccagc agggggtcat ggaggtaacc gaggctccgg aggaggcggc
121 ggcggcggag ggggtggtcg aggcggcagg ggccggcatc ccgggcacct gaaaggccgc
181 gaaatcggca tgtggtacgc gaaaaaacag gggcagaaga acaaggaagc ggagaggcaa
241 gagagagctg tagtacacat ggatgaacga cgagaagaac aaattgtaca gttactgaat
301 tctgttcaag cgaagaatga taaagagtca gaagcacaga tatcctggtt tgctcctgag
361 gatcatggat acggtactga agtttctact aagaacacac catgctcaga gaacaaactt
421 gacatccagg aaaagaagtt gataaatcaa gaaaaaaaaa tgtttagaat caggaacaga 481 tcatatattg accgagattc tgagtatctc ttgcaagaaa atgaaccaga tggaacttta
541 gaccaaaaat tattggaaga tttacaaaag aaaaaaaatg accttcggta tattgaaatg
601 cagcatttca gagaaaagct gccttcgtat ggaatgcaaa aggaattggt aaatttaatt
661 gataaccatc aggtaacagt aataagtggt gaaactggtt gtggcaaaac cactcaagtt
721 actcagttca ttttggataa ctacattgaa agaggaaaag gatctgcttg cagaatagtt
781 tgtactcagc caagaagaat tagtgccatt tcagttgcgg aaagagtagc tgcagaaagg
841 gcagaatctt gtggcagtgg taatagtact ggatatcaaa ttcgtctcca gagtcggttg
901 ccaaggaaac agggttctat cttatactgt acaacaggaa tcatccttca gtggctccag
961 tcagacccgt atttgtccag tgttagtcat atcgtacttg atgaaatcca tgaaagaaat
1021 ctgcagtcag atgttttaat gactgttgtt aaagaccttc tcaattttcg atctgacttg
1081 aaagtaatat tgatgagtgc aacattgaat gcagaaaagt tttcagaata ttttggtaac
1141 tgtccaatga tacatatacc tggttttacc tttccggttg tggaatatct tttggaagat
1201 gtaattgaaa aaataaggta tgttccagaa caaaaagaac acagatgcca gtttaagagg
1261 ggtttcatgc aagggcatgt aaatagacaa gaaaaagaag aaaaagaagc aatatataaa
1321 gaacgttggc cagattatgt aagggaactg cgaagaaggt attctgcaag tactgtagat
1381 gttatagaaa tgatggagga tgataaagtt gatctgaatt tgattgttgc cctcatccga
1441 tacattgttt tggaagaaga ggatggtgcg atactggtct ttctgccagg ctgggacaat
1501 atcagcactt tacatgatct cttgatgtca caagtaatgt ttaaatcagt taaccagaca
1561 caggtgttta aaagaacccc tcctggtgtt cggaaaatag taattgctac caacattgcg
1621 gagactagca ttaccataga tgatgtcgtt tatgtgatag atggaggaaa aataaaagag
1681 acgcattttg atactcagaa caatatcagt acaatgtccg ctgagtgggt tagtaaagct
1741 aatgccaaac agagaaaagg tcgagctgga agagttcaac ctggtcattg ctatcatctg
1801 tataatggtc ttagagcaag tcttctagat gactatcaac tgccagaaat tttgagaact
1861 cctttggaag aactttgttt acaaataaag attttaaggc taggtggaat tgcttatttt
1921 ctgagtagat taatggaccc accatcaaat gaggcagtgt tactctccat aagacacctg
1981 atggagctga acgctttgga taaacaagaa gaattgacac ctcttggagt ccacttggca
2041 cgattacccg ttgagccaca tattggaaaa atgattcttt ttggagcact gttctgctgc
2101 ttagacccag tactcactat tgctgctagt ctcagtttca aagatccatt tgtcattcca
2161 ctgggaaaag aaaagattgc agatgcaaga agaaaggaat tggcaaagga tactagaagt
2221 gatcacttaa cagttgtgaa tgcgtttgag ggctgggaag aggctaggcg acgtggtttc
2281 agatacgaaa aggactattg ctgggaatat tttctgtctt caaacacact gcagatgctg
2341 cataacatga aaggacagtt tgctgagcat cttcttggag ctggatttgt aagcagtaga 2401 aatcctaaag atccagaatc taatataaat tcagataatg agaagataat taaagctgtc
2461 atctgtgctg gtttatatcc caaagttgct aaaattcgac taaatttggg taaaaaaaga
2521 aaaatggtaa aagtttacac aaaaaccgat ggcctggttg ctgttcatcc taaatctgtt
2581 aatgtggagc aaacagactt tcactacaac tggcttatct atcacctaaa gatgagaaca
2641 agcagtatat acttgtatga ctgcacagag gtttccccat actgtctctt gttttttgga
2701 ggtgacattt ccatccagaa ggataacgat caggaaacta ttgctgtaga tgagtggatt
2761 gtatttcagt ctccagcaag aattgcccat cttgttaagg aattaagaaa ggaactagat
2821 attcttctgc aagagaagat tgaaagtcct catcctgtag actggaatga cactaaatcc
2881 agagactgtg cagtactgtc agctattata gacttgatca aaacacagga aaaggcaact
2941 cccaggaact ttccgccacg attccaggat ggatattaca gctga
Differential Cellular Localization of RHAU and RHAUΔU
The cellular localization of RHAU is examined by expressing HA-RHAU and HA- RHAUΔ14 in HeLa-Tet-Off (TO) cells, followed by immunocytochemistry. Levels of expression of both RHAU isoforms are examined by Western blotting using an anti-HA epitope antibody. Using the same antibody for indirect immunofluorescence, we found that the two RHAU isoforms displayed distinct subcellular localization. RHAU and RHAUΔ14 are preferentially localized to the nucleus and cytoplasm, respectively. However, partitioning of cellular localization between the nucleus and cytoplasm was not exclusive, as the staining of full length HA-RHAU was also obvious in the cytoplasm. Both isoforms are excluded from the nucleoli (data not shown). These results suggest that RHAU contains a putative nuclear localization sequence of at least 14 amino acids.
Overexpression of RHAU and RHAUΔ14 Enhances AREuPA-mRNA Decay
Because RHAU was identified as an AREuPA-associated factor, we examined its effect on the stability of a β-globin chimeric mRNA that harbored the AREuPA in its 3' UTR. This β-globin- AREuPA (β-glo-ARE) reporter gene, under the control of a tetracycline-responsive element, is stably expressed in HeLa-TO cells and the de novo synthesis of β-glo-ARE mRNA could be selectively terminated by addition of doxycycline, a tetracycline derivative. β-glo-ARE mRNA has a half-life (t1/2) of approximately 3 hours in vector-transfected cells (data not shown). Overexpression of HA-RHAU modestly reduced the stability of β-glo-ARE mRNA (ti/2 approx. 2 hours) (data not shown). Notably, a more significant reduction of β- glo-ARE mRNA stability (3-fold) is observed when the cytoplasmically localized HA-RHAUΔ14 is overexpressed (data not shown). Overexpression of HA-RHAU and HA-RHAUΔU in HeLa-TO cells stably expressing a β-globin wild-type mRNA that has an extrapolated half-life of more than 15 hours did not affect the stability of this message (data not shown). However, we cannot rule out the possibility that a higher level of RHAU overexpression could affect the high stability and abundance of wild-type β-globin mRNA. Overexpression of the RHAU isoforms did not affect the stability of ribosomal RNA or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (data not shown). Unlike commonly used HeLa cells, HeLa-TO cells express a substantial level of endogenous uPA and uPAR (urokinase-type plasminogen activator receptor) mRNAs. HA-RHAU overexpression in these cells significantly enhances the destabilization of uPA mRNA (ti/2 approx. 2.2 hours in vector-transfected cells compared with Xy2 approx. 1.2 hours in HA-RHAU-transfected cells). Strikingly, HA-RHAU overexpression has no effect on the stability of the ARE-containing uPAR mRNA (ti/2 approx. 3 hours in both vector- and HA-RHAU-transfected cells). This suggests that the specificity of RHAU may be restricted to a limited number of mRNAs. We conclude from these experiments that overexpression of RHAU specifically accelerates the decay of mRNAs containing the AREuPA but not those without.
Recombinant GST-RHAU Enhances RNA Deadenylation and Decay in Vitro
To further our observations in vivo, the effect of RHAU on ARE-mRNA decay in vitro was examined. We used a previously described cell-free system that supports ARE-stimulated RNA deadenylation and decay (Ford et al., 1999, Genes Dev. 13: 188-201). Purified, recombinant GST or GST-RHAU are incubated with HeLa S100 proteins and32P-labeled, capped and polyadenylated RNA substrates derived from 140 nucleotides of the rabbit-globin 3' UTR. Consistent with in vivo data, the presence of the AREuPA stimulated the decay of the β-globin (glo-ARE-A60) RNA substrate in vitro (data not shown). In this system, addition of GST-RHAU, but not GST, further enhanced the ARE- stimulated decay (data not shown). Interestingly, while fully deadenylated intermediates are not observed in control reactions, substrates incubated in the presence of GST-RHAU produced decay intermediates that are almost completely deadenylated (data not shown). To rule out the possibility that GST- RHAU (or a copurified, trace contaminant) may have intrinsic RNase activity, GST-RHAU is incubated with glo-ARE-A60 RNA for 60 minutes in the presence or absence of HeLa S100 proteins. In contrast to the deadenylation and decay observed in the presence of S100 proteins, the RNA substrate is neither deadenylated or degraded in the absence of HeLa S100 proteins (data not shown). We found that GST-RHAU does not significantly stimulate the decay of a non-ARE-containing (glo-A60) RNA substrate and deadenylation was only slightly stimulated (data not shown). GST- RHAUΛU produced similar, if not identical, results in the assays described above (data not shown). These results complement in vivo data with respect to AREuPA-directed mRNA decay and its enhancement by RHAU.
Downregulation of RHAU Increases the Stability of Endogenous uPA mRNA
We asked if depletion of RHAU could exert an effect opposite to its overexpression and stabilize labile mRNAs in vivo. Data indicate that when HeLa-TO cells are transfected with an siRNA that targets both RHAU and RHAUΔU mRNAs, endogenous RHAU and RHAUΔU protein levels are effectively depleted by greater than 70% of normal levels after 48 hours (data not shown). The specificity of RHAU depletion in these cells is confirmed by probing the same blot with an antibody against NFAR1 (data not shown). In RHAU- depleted cells, endogenous uPA mRNA is stabilized approximately 2-fold (ti/2 approx. 4 hours) compared to cells transfected with a control siRNA (ti/2 approx. 2.2 hours) or mock transfected (W2 approx. 1.9 hours) (data not shown). Consistent with observations in HA-RHAU overexpression experiments, downregulation of RHAU has no significant effect on the stability of uPAR mRNA compared to control-transfected cells (t|/2 approx. 2 hours) (data not shown).
The ATPase Activity of RHAU is Required for Enhancement of mRNA Destabilization In Vivo and In Vitro
The ATPase activity of DExH/D RNA helicases are known to be stimulated by nucleic acids. We tested whether RHAUΔU (results described below are equally applicable to RHAU, data not shown) has ATPase activity in vitro and if this activity is enhanced in the presence of dsDNA, dsRNA, tRNA or homopolymeric RNAs. Normalized to GST control reactions, [γ-32P] ATP hydrolysis by recombinant GST- RHAUΛ14 is modestly increased (approx. 2 fold) with tRNA, dsDNA, dsRNA and poly (A1C), but markedly stimulated (> 4-fold) by poly(U) (data not shown). This finding is interesting since the majority of AREs, including the AREuPA , are in fact stretches of uridylates interrupted by adenylates. To obtain an ATPase-defective mutant of GST- RHAUΔ14, the invariant glutamate at position 335 (E335) in the DEIH motif was substituted with alanine (E335A). In ATPase assays, in the presence of poly(U), where GST-RHAUΔ14 is titrated giving submaximal ATPase activity with 200 ng, the same amount of GST- RHAUΔ14-E335A shows no ability to hydrolyze ATP (data not shown). To test if the ATPase activity of HA- RHAUΔ14 is important for its ability to enhance AREuPA -mediated destabilization, HA- RHAUΔ14 and RHAUΔ14-E335A are transiently transfected in HeLa-TO-β-globin- AREuPA cells. Similar expression levels are confirmed for both wild-type and mutant versions of HA- RHAUΔ14 (data not shown). Consistent with results already shown, HA- RHAUΔ14 significantly enhanced the destabilization of β-globin-ARE-mRNA (data not shown). In contrast, HA-RHAUΔ14-E335A did not enhance β-globin-ARE mRNA decay but even had a moderate, but not significant, negative effect on AREuPA-mediated mRNA turnover (data not shown). Parallel effects are observed in cell-free RNA decay assays where GST-RHAUΔ14, but not GST-RHAUΔ14-E335A, could enhance ARE-RNA deadenylation and decay (data not shown). We conclude that E335 in the DEIH motif of RHAU is essential for its ATPase activity and that this activity is required for the function of RHAU in promoting mRNA deadenylation and decay.
RHAU Physically Interacts with the Exosome and Poly A Specific Ribonuclease (PARN) and Exhibits RNA-Dependent Interaction with NFAR1 and HuR
Because a putative yeast DExH RNA helicase was previously implicated as a cofactor for exosome-mediated mRNA decay (Jacobs et al., 1998, EMBO J. 17: 1497-1506), we postulated that RHAU may similarly serve as a cofactor for exosome-mediated ARE-mRNA decay in mammalian cells. Coimmunoprecipitation experiments are performed using lysates from HeLa cells transfected with HA-RHAU. lmmunoprecipitations using anti-HA antibodies but not an isotype-matched IgG control antibody coprecipitated both human exosome components hRrp40p and PM-ScHOO (data not shown). Notably, these interactions are insensitive to RNase treatment (data not shown). The amount of coprecipitated hRrp40p and PM-ScMOO are significantly increased using RNase- treated lysates, in part because more HA-RHAU is immunoprecipitated (data not shown). This suggests that a fraction of cellular RHAU may have been sequestered into mRNA complexes and is not accessible for antibody binding, but more importantly, this experiment demonstrates a physical, RNA- independent interaction between RHAU and the exosome. In the reverse experiment, immunoprecipitations of the exosome components hRrp40p or PM- ScH 00, in the presence of RNases, coprecipitated HA-RHAU, whereas a control immunoprecipitation using normal rabbit sera failed to do so (data not shown). In the same experiment, using anti-PARN antisera, we detected substantial coprecipitation of HA-RHAU but not the exosome components (data not shown). Immunoprecipitations using lysates from vector-transfected cells are performed as negative controls. These results are reproduced with lysates from HA- RHAUΔ14-transfected cells but the interaction between HA- RHAUΔ14 and PM- ScHOO is reduced (data not shown).
Because recombinant RHAU proteins do not bind the AREuPA in gel mobility shift assays (data not shown), we suspected that its association with the AREϋPA during ARE-affinity chromatography and its specificity for enhancing the decay of AREuPA-RNAs is a result of indirect protein-protein interactions, likely with HuR or NFAR1, or both. This is confirmed by a coimmunoprecipitation assay using antibodies against NFAR1 and HeLa lysates in the presence or absence of RNases. HuR and a small amount of endogenous RHAU coprecipitated with NFAR1 , but only in the presence of RNA (data not shown). We note that the interaction between NFAR1 and HuR is also dependent on RNA. Similarly, a GST pull-down assay reveals that NFAR1 and RHAU is coprecipitated with GST- HuR from lysates not treated with RNases but this interaction is not detected using RNase-treated lysates (data not shown). Interestingly, we also find protein kinase R (PKR) coprecipitating in the GST-HuR pull-down (data not shown). PKR is an interferon- and dsRNA-activated kinase known to play a major role in the antiviral response and induction of apoptosis. Importantly, PKR is known to interact with and phosphorylate DRBP76/NFAR1 in vitro.
The results presented above indicate that RHAU is a DExH RNA helicase and is an AREuPA - associated factor. It was demonstrated that RHAU plays a role in AREυPA- mRNA deadenylation and decay and this specificity may be linked to its RNA-dependent interaction with the AU binding proteins (AUBPs) HuR and NFAR1. Additionally, we demonstrated a physical association between RHAU and the human exosome, and the deadenylase PARN (Dehlin et al. 2000, EMBO J. 19: 1079-1086). A causal role for RHAU in AREuPA-mRNA metabolism is confirmed by overexpression and downregulation experiments. Overexpression of RHAU enhanced the degradation of uPA and reporter β-globin-AREuPA mRNAs, but not GAPDH or a non-AREuPA-containing β-globin mRNA, while its downregulation by RNA interference stabilized endogenous uPA mRNA. Furthermore, recombinant RHAU in cell-free RNA decay reactions accelerated the decay of an AREuPA-containing RNA but not a non-ARE RNA substrate. Importantly, it was demonstrated that the enhancement of AREuPA-mRNA- destabilization by RHAU is dependent on its ability to hydrolyze ATP.
It is surprising that while overexpression and depletion of RHAU destabilized and stabilized, respectively, uPA mRNA, it had no effect on the ARE-containing uPAR mRNA in vivo. Like the AREuPA, the class I ARE of uPAR mRNA is an effective mRNA-destabilizing element (Wang et al., 1998, PNAS USA, 95: 6296-6301). Moreover, we have shown that HuR specifically bind both ARE sequences and stabilize RNAs containing these AREs in vivo and in vitro (Tran et al., 2003, MoI. Cell. Biol. 23: 7177-7188). Our present data suggests that the functional specificity of RHAU may be limited to a very select group of ARE-containing mRNAs.
While the HeLa S100 extracts we prepared and used for cell-free RNA deadenylation/decay assays is not efficient in deadenylation over the experimental time course, however, in the presence of GST-RHAUΔ14, deadenylation of the RNA substrate at 60 minutes is almost complete (data not shown). Importantly, our data indicate that RHAU interacts with PARN1 a poly(A) specific exoribonuclease that plays a role in mRNA deadenylation in vivo and in HeLa extracts. Taken together, these results strongly suggest that RHAU can stimulate the activity of PARN leading to the enhancement of RNA deadenylation and subsequent decay.
The role of the yeast exosome in deadenylation-dependent, 3'-to-5' mRNA decay was proposed to be modulated by the DExH protein Ski2p (Jacobs et al., 1998, EMBO L. 17: 1497-1506). Data presented here indicate that RHAU is associated with two components of the human exosome. This association is more pronounced in RNase-treated cell extracts partly because more HA-RHAU could be immunoprecipitated. This may suggest that RHAU is not a core component of the exosome, but rather, their interaction is dynamic. Interestingly, we find partial and complete disruption of interaction between the exosome subunits hRrp40p and PM-ScH 00, respectively, when these proteins are immunoprecipitated from HA-RHAU-expressing cell extracts. We take this result to mean that RHAU binding may have displaced one or more subunits from the exosome, suggesting a mechanism for the allosteric activation of the exosome. It is essential to investigate the exosome subunit to which RHAU is bound and the regions of RHAU responsible for the interaction. A putative DExH RNA helicase closely related to yeast Ski2p has been coprecipitated with the human exosome, but its role in mRNA degradation has not been determined (Chen et al., 2001 , Cell 107: 451-464).
In mammalian cell extracts where 3'-to-5' mRNA turnover is the predominant pathway, the exosome is functionally coupled to a scavenger decapping activity that follows mRNA deadenylation and decay. It has previously been proposed that this degradation-dependent mRNA decapping activity is complexed to a subset of exosome components specific for 3'-to-5' mRNA decay. No link has been found between RNA helicases and degradation-dependent mRNA decapping, but on the prior assumption that many established in vitro decay extracts are competent for exosome-mediated, 3'-to-5' mRNA decay (Chen et al., 2001 Cell 107: 451-464; Wang and Kiledjian, 2001 , Cell 107: 751-762), we propose that RHAU may be complexed to a pool of cellular exosomes dedicated to the 3'-to-5' degradation of selected mRNAs.
An unsolved question is how RNA helicases, which by their functional nature should translocate rapidly along and unwind dsRNA or disrupt RNA-protein interactions, can specifically recognize their RNA substrates. The demonstration that an RNA helicase can directly interact with a specific RNA sequence is lacking, and it is widely believed that substrate specificity is mediated indirectly by protein-protein interactions. Our data supports the idea that the specificity of RHAU toward selective degradation of AREuPA-mRNA is mediated by its RNA- dependent interaction with the AUBPs HuR and NFAR1. This interaction is demonstrated by ARE-affinity chromatography, NFAR1 coimmunoprecipitation and GST-HuR pull down experiments. Because only a small fraction of RHAU associated with NFAR1 or HuR and because we could not detect NFAR1 or HuR in RHAU immunoprecipitates, it is likely that their association in the cell is highly dynamic and transient. Furthermore, in gel mobility shift experiments, recombinant RHAU proteins do not directly interact with the AREuPA. A component of the human exosome, PM-SCI75, was shown to have ARE binding activity, however, it is unlikely that RHAU is bound to the AREuPA via the exosome because the stoichiometric presence of exosomal proteins are not detected in the AREuPA-bound fractions. Confirmation will come from future biochemical and/or structural studies of RHAU and the exosome.
We consider it plausible that different RNA helicases are associated with ribonucleases (like the exosome) and specific RNA binding protein complexes to entail target specificity of RNA degradation. Consistent with this idea, others have described a protein complex that contains, among other components, a double-stranded RNA binding protein, the RNase Ill-related Dicer and a DExH RNA helicase that is required to direct RNA interference in Caenorhabditis elegans. Similarly, the DExH protein Suv3p has been shown to be a component of a yeast mitochondrial 3'-to-5' exoribonuclease activity that is required for degradation of group I intron RNAs. Thus, we propose that ATP-dependent RNA helicases serve as "molecular motors" coupled to specific RNA binding proteins to drive the mechanics of complex RNA remodeling/decay reactions.
Based on biochemical evidence of RNA unwinding activity attributed to some DExH/D RNA helicases, and on strong sequence conservation of ATP binding and helicase motifs, it is widely assumed that DExH/D proteins possess the ability to unwind dsRNA. However, because the existence of long dsRNA in the cell is rare and because most cellular RNAs are complexed with proteins, the primary function of DExH/D RNA helicases may be to disrupt RNA-protein interactions. This idea is supported by strong biochemical evidence that demonstrated the displacement of a high-affinity interaction between U1A protein and its cognate RNA binding site by the vaccinia virus DExH protein NPH-II (Jankowsky et al., 2001 , Science 291: 121-125).
We propose a working model suggesting that the primary function of RHAU is to disrupt RNA-protein interactions, for example, HuR and/or NFAR1 (mRNA- stabilizing proteins) from the ARE, and in doing so, facilitate ARE-mRNA decay. According to this model, first RHAU associated with PARN (with or without the exosome) recognizes an ARE-mRNA substrate and stimulates RNA deadenylation. At this stage, the exosome is still inactive. Upon completion of poly(A)-tail removal, RHAU may then begin to disrupt the interaction between HuR/NFAR1 and the ARE. Recognition of AUBPs or the protein-free ARE strongly stimulates the ATPase activity of RHAU which in turn activates the exosome. Simultaneously, the exosome may be recruited to the ARE by mRNA- destabilizing AUBPs to begin the rapid exonucleolytic decay of the RNA body. Utilizing the cell-free decay system, we can begin to address the mechanistic details regarding the involvement of RHAU in mRNA degradation.
The data presented above have been reported in Tran, H, Schilling, M, Wirbelauer, C, Hess, D, and Nagamine, Y, 2004, MoI. Cell 13: 101-11 , the content of which is hereby incorporated by reference in its entirety.
EXAMPLE 2
RHAU is Important for the Survival of Cancer Cells: RHAU Depletion in Tumor and Non-Tumor Derived Cell Lines
The effect of RHAU depletion on the survival on cells derived from tumor and non-tumor sources was assayed utilizing RNAi according to the methods provided below. Experimental Methods
Cell culture
HeLa Tet-off cells (Clontech) are maintained in Dulbecco's Modified Eagle's
Medium (DMEM, Gibco BRL) supplemented with 10% (v/v) fetal calf serum (ANIMED; BioConcept, Allschwil, Switzerland), 0.2 mg/mL streptomycin, 50 units/mL penicillin andlOO ng/ml G-418 (Invitrogen) at 37° C in a humidified 5% CO2 incubator. PNT-2 and PC3 cells are maintained in RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum and the antibiotics described above, except G-418. MCF-10A cells are maintained in DMEM supplemented with 10% (v/v) horse serum and the antibiotics described above, except G-418.
RNA transfection
The 21-mer oligoribonucleotide sequence of siRNAs used to target RHAU mRNA are as follows: sense, 5'-GGG AAC UGC GAA GAA GGU AUU-3' (SEQ ID NO:5) and antisense, 5'-UAC CUU CUU CGC AGU UCC CUU-3' (SEQ ID NO:6). These sequences contain 3' UU overhangs and target both RHAU isoforms. siRNA sequences targeting NFAR1 are as follows: sense, 5'-AAC UUC UCC CGC CUC UUG UAA-31 (SEQ ID NO:10) and antisense, 5'-UUA CAA GAG GCG GGA GAA GUU CGC CUC UUG UAA-31 (SEQ ID NO:11). To ensure gene specificity, designed RNA oligoribonucleotide sequences are searched against the GenBank/EMBL database using the BLAST program. siRNAs were purchased from Xeragon (Huntsville, Alabama).
A day before transfection of small interfering RNA (siRNA) duplexes, cells are seeded in 35-mm dishes at 1.4 x 105 cell/dish. The next morning, siRNAs are introduced into cells using OligofectAMINE reagent (Life Technologies) according to the manufacturer's intructions, with 10 μ\ of 20 μM siRNA and 3 μl of transfection reagent/dish. Cells are photographed and collected for protein/RNA analysis according to conventional methods two days after transfection.
Results
Depletion of RHAU appears to significantly affect the survival of tumor-derived cell lines suggesting that it is essential for cancer cell biology. PNT-2 and MCF10A are cell lines derived from normal prostate and breast tissue, respectively. The ability of these cells to proliferate and repopulate the culture dish to confluency was not affected 48 hours after transfection with siRNA against RHAU (siRHAU) to downregulate endogenous RHAU protein levels. Similarly, cells transfected with siNFARI showed no effects on cell growth compared with a buffer control. In contrast, PC3 (prostate cancer) cells and HeLa (cervical adenocarcinoma) cells showed signs of apoptotic cell death 48 hours after transfection with siRHAU. With siNFARI transfection, these cells show impaired proliferation but no significant cell death. Thus, depletion of RHAU appeared to significantly affect the survival of tumor-derived cell lines, suggesting that it is essential for cancer cell biology. If the cell death observed in RHAU- depleted cells is caused by apoptosis, then RHAU may have an anti-apoptotic function or be connected to such cellular mechanisms. Future studies to verify and extend these studies are warranted and may be greatly assisted by targeted gene inactivation of RHAU in mice. As RHAU specific siRNA preferentially suppress the growth of transformed cells it is possible that binding partners of RHAU are different in transformed cells compared to non-transformed cells. Identification of transformed cell-specific partners then may provide additional targets for tumor therapy. In order to identify such RHAU-binding partners, antibodies against RHAU can be used in co-precipitation analysis using cell extracts derived from transformed and non-transformed cells. Binding partners can then be identified by SDS-polyacrylamide gel electrophoresis followed by mass spectrometry. For example, antibodies against HA and GST can be used after these cells are transfected with expression vectors for HA-tagged and GST- tagged RHAU.
EXAMPLE 3
Additional Genetic and Biochemical Studies to Further Characterize the
Mechanism of Action of RHAU
In addition to the regulation of mRNA stability in the cytoplasm, the mainly nuclear localization of RHAU indicates other possible functions for RHAU in the cell. Further characterization may include genetic studies to determine whether RHAU is essential for the survival of the cell or organism and how its deletion affects cellular function and embryonic development. For example, RNAi- mediated RHAU downregulation may be performed in cultured cells of various types using RHAU-specific siRNA according to conventional methods. In previous experiments (see Example 2), the prostate and breast cancer cell lines PC3 and MDA-MB-231, respectively, died 4 days after RHAU downregulation, while their normal counter parts PNT-2 and MCF-1 OA, respectively, did not. This suggests that RHAU is essential for the survival of tumor cells but is dispensable for non-transformed cells. To further explore this possibility, studies using a RHAU expression vector with silent mutations at the siRNA-targeting site may be performed and overexpression of RHAU with silent mutations simultaneously with or before RHAU-specific siRNA transfection should alleviate the growth inhibitory effect of siRNA in tumor cells if the inhibition described above is due to RHAU downregulation. These "knockdown-in" studies may be performed according to conventional methods and as previously reported in Kisielow, M et al., 2002, Biochem. J. 363: 1-5. Patterns of global mRNA expression in MDA-MB- 231 or PC3 cells before and after RHAU downregulation may be compared by means of an Affymetrix GeneChip Array using Human Genome U133 Set. mRNAs exhibiting a greater than twofold difference, either positively or negatively, will be selected and reconfirmed individually by Northern blot hybridization. Confirmed mRNAs may then be characterized for regulation of stability by RHAU and for the presence of AREs or common features in the 3' UTRs. The identification of these mRNAs will provide useful insight about the mechanism by which RHAU affects tumor cell growth.
Possible non-specific effects of siRNA reactions may be controlled for by preparing HeLa cell lines in which RHAU-specific siRNA can be induced by tetracycline according to methods known in the art which circumvent the lipid- mediated transfection step (van de Wetering, M et al., 2003, EMBO Rep. 4: 609- 615). A HeLa cell line stably expressing a tetracycline-suppressible repressor is available (van de Wetering, M et al., 2003, EMBO Rep. 4: 609-615). The patterns of global mRNA expression in this cell line before and after RHAU- specific siRNA expression may be compared as outlined above.
Data indicate that Northern blot analysis of various mouse tissues shows ubiquitous expression of RHAU and especially high expression in brain, thymus and testis (Takahashi, Y, 1992, Prog. Neurobiol 38: 523-569). The complexity of mRNAs expressed in brain is very high, suggesting a high turn-over rate. Thus, it will be interesting to investigate specific effects of RHAU in brain and experiments to knock out the RHAU gene globally and specifically in brain, may be performed.
Global targeting of the RHAU gene
The RHAU gene is composed of 25 exons with the Met start codon residing in the first exon. As the first intron includes no open reading frames that could be linked to a downstream coding region without hitting termination codons, it is very unlikely that deletion of the first exon would give rise to an active, truncated RHAU protein. Therefore, a targeting (KO1) vector that replaces the first exon of the murine RHAU gene with a neomycin-resistance gene may be constructed according to methods familiar to one of skill in the art. This vector contains 3 kb and 7 kb of the 5' and 3' flanking regions, respectively, of the first exon of the RHAU gene. The vector may be injected into embryonic stem cells and correctly targeted cells selected by conventional PCR analysis and further processed to derive KO mice according to conventional methods as can be found in, e.g., Joyner, AL, ed., Gene Targeting - A Practical Approach, 2nd ed., Oxford University Press.
Conditional knockout of the RHAU gene in the brain
To achieve brain-specific targeting of the RHAU gene using a Cre-LoxP system (Dymecki, S, p. 37-100, In: Joyner, AL, ed., Gene Targeting - A Practical Approach, 2nd ed., Oxford University Press), a targeting vector (KO2) can be constructed in which exon 8 of the RHAU gene is flanked by the LoxP sequences. Exon 8 codes the DExH box, which is essential for RHAU ATPase activity and for stimulating ARE-mRNA decay. Utilizing methods familiar to one of ordinary skill in the art, the LoxP-RHAU mice may be crossed with mice expressing Cre in brain or other tissues under tissue-specific promoters.
In addition to the RNA helicase core region in the middle of the RHAU, which is highly conserved among many DExH-box RNA helicases, RHAU contains unique amino-terminal and carboxyl-terminal regions that are not shared with other RNA helicases. These regions are conserved as highly as the RNA helicase core regions between human and mice, suggesting that they have important functions. To reveal the specific functions associated with these regions, conventional GST- fusion protein pull-down assays may be used to search for proteins interacting with these regions. Conventional methodologies may be used to fuse the amino terminal or carboxyl terminal region of RHAU with GST in a bacterial expression vector in which expression is under control of IPTG. The hybrid protein may then be coupled to glutathione-Sepharose beads, which may then be used for pulldown assays using nuclear or cytoplasmic extracts from HeLa cells. Specifically bound proteins can be separated by 2-D gel electrophoresis and identified by mass spectrometry. Specific binding proteins identified in this analysis will be tested for their interaction with RHAU both in a cell-free system using purified samples and in the cell using appropriate expression vectors. Once their interaction with RHAU is established, these proteins may be studied with regard to localization in the cell and indispensability for cell growth.