| DHX8 | |||||||||||||||||||||||||||||||||||||||||||||||||||
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| Identifiers | |||||||||||||||||||||||||||||||||||||||||||||||||||
| Aliases | DHX8, DDX8, HRH1, PRP22, PRPF22, DEAH-box helicase 8, Dhr2 | ||||||||||||||||||||||||||||||||||||||||||||||||||
| External IDs | OMIM:600396;MGI:1306823;HomoloGene:3628;GeneCards:DHX8;OMA:DHX8 - orthologs | ||||||||||||||||||||||||||||||||||||||||||||||||||
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| Wikidata | |||||||||||||||||||||||||||||||||||||||||||||||||||
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DEAH-box helicase 8, is aprotein that in humans is encoded by theDHX8gene. This protein is member of theDEAH box polypeptide family. The main characteristic of this group is their conserved motif DEAH (Asp-Glu-Ala-His).[5] A wide range ofRNA helicases belongs to this family. Specifically, DHX8 acts as anATP-dependent RNA helicase involved insplicing and the regulation of the releasing of spliced mRNAs fromspliceosomes out of the nucleus.[6] Published studies have shown the consequences of DHX8mutations, some of them are critical for biological processes such ashematopoiesis and are related to some diseases.[7][8]
Protein DHX8 is part of a protein complex called spliceosome, which is in charge ofpre-mRNA splicing. The spliceosome has eight major functional states, each with distinct composition and structure; five of the eight states have been structurally characterized.[10]
DHX8 have different domains: aS1 RNA binding domain (DEAD/DEAH box), an helicase conservedC-terminal domain, helicase associated domain (HA2), and anoligonucleotide/oligosaccharide-binding (OB)-fold, each joined byintrinsically disordered regions.[11]
There are some regions of the protein which are very important for its activity, like R620 and the hook-loop and hook-turn regions. Also, DHX8Δ547 is the catalytically active core of the protein DHX8. It is made of twoRecA domains and the C-terminal WH, ratchet-like and OB-fold domains and theN-terminal region.[9]
The total weight of the DHX8 structure is 156580.13 Da.
Secondary structure:
DHX8 is localized in the cellular nucleus and stimulated upon RNA presence. This protein is a component of the spliceosome, so it takes part in pre-mRNA splicing.[9] Splicing is the process of joiningexons from primary transcripts of messenger RNA and the elimination ofintron sequences, by means of a spliceosomal mechanism, so that the mRNA produced is the one without introns, consisting exclusively of the joined exons.[12] Splicing finishes with the spliceosomal complex disassembly and the ATP-dependent liberation of the resulting mature RNAs to the outer of the nucleus.[13] Spliceosome requires conformational changes to be able to catalyze splicing reactions and the later mature mRNA releasing to the outer of the nucleus. One of the ATP-dependent helicase needed for these conformational changes is DHX8. Furthermore, DHX8 plays a key role in the releasing, facilitating the nuclear export of spliced mRNA. Protein characterization has shown that DHX8 has a binding preference foradenine-rich RNA. This binding is followed byATP hydrolysis and thus,ADP release.[9]
DHX8 has multiple molecular functions like ATP-binding by, selective and non-covalent interactions with the coenzyme and enzyme regulator adenosine 5’ triphosphate.[14] Also identical protein binding (creates a similar type of interactions as described above but with other proteins), RNA binding and RNA helicase activity, based on catalysis of the reaction that unwinds an RNA helix:[15]
ATP + H2O = ADP + phosphate[16]
DHX8 protein has its main focuses of expression in the brain, especially in thecerebellum where it can be found mostly inPurkinje cells. Other important are theprostate and thegallbladder, which are zones where the polypetide is found also highly expressed.
DHX8 protein is not expressed in all tissues and organs significantly, some clear examples are the bone marrow or the soft tissue (Peripheral nerve), where we do not have enough quantity of the protein to be representative.[17]
Assays in enzymology for the biochemical characterization of proteins need high concentrations of the protein of interest and its protocols should be efficient, simple and cost-efficient to ensure a successful purification. One example of a purification approach for DHX8 is via aprotein tag called GST-His grafted onto DHX8 protein is used.
The N-terminal Glutathione Sepharose TAG (GST) and C-termianlHis-tag, also known as GST-His is a 29 kDa tag which allows small-scale affinity purification for recombinant proteins. This method is based on two different tags flanking the two extremes of the protein. However, it might influence physiological properties of the protein and thus, empirical testing is required for each case.[18]
DHX8 constructs are generated by PCR cloning using restriction enzymes. To generateHis6GST-DHX8Δ54 (hexahistidine-GST), the coding sequence for residues A548 to R1220 is inserted into a version of "pFastBac", a specific vector kit of Thermo Fisher Scientific,[19] modified to encode an N-terminal His6GST-tag followed by an HRV 3C protease (recombinant restriction-grade protease) cleavage site. HRV 3C is a highly purified recombinant 6XHis-fusion protein, that recognizes the same cleavage site as the native enzyme. Both, vector and insert, are digested withNdeI andEcoRI restriction enzymes.
To create full-length DHX8 (fl-DHX8-His6) and DHX8Δ547-His6, PCR primers are designed, so that a His6-tag is fused to the C-terminus of DHX8. The resultant PCR amplicons encoding fl-DHX8 (M1 to R1220) or DHX8Δ547 (A548 to R1220) are inserted into the "pFBDM" vector[20] downstream of the polyhedrin promoter. Both, vector and insert, are digested withBamHI andNotI restriction enzymes.[9]
