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.2000 Oct;20(20):7463-79.
doi: 10.1128/MCB.20.20.7463-7479.2000.

Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein

Affiliations

Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein

V Markovtsov et al. Mol Cell Biol.2000 Oct.

Abstract

Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, we characterized the binding of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. We also identified a new 60-kDa tissue-specific protein that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins.

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Figures

FIG. 1
FIG. 1
Structure and composition of the DCS complex. (A) nPTBcross-links to the CUCUCU element of the DCS RNA. The DCSRNA sequence is shown at the top. Modified bases are shown as hollowletters. Site-specific labeling of proteins binding to the DCS RNA isshown below the sequence. Cross-linking in the ASP40 fraction of WERI-1(lane 1) or HeLa (lane 2) extract and in total HeLa (lane 3) or WERI-1(lane 4) nuclear extracts is shown. Positions and sizes of themolecular weight standards (in thousands) are indicated on the right.(B) Immunoprecipitation of the proteins binding to the DCS RNA in theWERI ASP40 fraction. Antibodies used were anti-SM Y12 (lane 2),anti-KSRP (lane 3), anti-FBP (lane 4), and anti-PTB (lane 5). (C)Probing hnRNP H, hnRNP F, KSRP, FBP, and nPTB binding sites on the DCSRNA by site-specific labeling. Modified (hollow) and radiolabelednucleotides are shown below the DCS RNA sequence. Cross-linking wasdone in the ASP40 fraction of WERI-1 (lanes W) or HeLa (lanes H)nuclear extract. (D) DCS RNA mutants used in the cross-linkingexperiments. Positions of 4-thioU modifications are indicated asU8 and U18 above the wild-type (WT) sequence,and the mutations are indicated below. (E) Effect of mutations in DCSRNA on the cross-linking of proteins to positions U18 (top)and U8 (bottom) of the DCS in the ASP40 fraction of WERI-1extract (lanes W) or HeLa extract (lanes H).
FIG. 2
FIG. 2
Purification of proteins bound to the DCS RNA or anunrelated RNA in the WERI-1 or HeLa extracts using RNA affinitychromatography. (A) Coomassie blue-stained gels displaying fractionsfrom the DCS RNA affinity column (lanes 1 to 6 and lanes 8 to 13) or anunrelated RNA affinity column (lanes 15 to 20). Proteins identified byMS are indicated by arrows. Lanes 7, 14, and 21 contain proteinmarkers. (B) Western blot analysis of the same fractions. MAb104monoclonal antibody was used for identification of the SR proteins. Thepositions of the SC35 and ASF/SF2 (SRP 35), SRp55, and SRp75 proteinsare indicated by arrows.
FIG. 3
FIG. 3
Purification of human nPTB. (A) Purification scheme.Coomassie blue-stained gels of PTB- and nPTB-containing fractions fromthe HiTrap Blue column are shown below the scheme. (B) Coomassie bluestaining of an SDS–10% polyacrylamide gel containing purified WERI-1nPTB and HeLa PTB. (C) nPTB peptide sequences identified by Edmandegradation (left) and corresponding human PTB-1 peptides (right).Differences between two proteins are shown by hollow letters.
FIG. 4
FIG. 4
Human nPTB cDNA sequence. The NLS sequences are shaded.The four RRM domains are boxed. Sequenced peptides are in bold. Theasterisk indicates the stop codon.
FIG. 5
FIG. 5
Comparative analysis of the nPTB amino acid sequence.(A) Structural organization of the nPTB RRMs. RNP1 and RNP2 consensussequences for the typical RRM and for the nPTB RRMs are shown at thetop. The domain secondary structure is schematically shown below theRRM sequences. Residues identical in at least two RRMs are shaded. (B)Comparison of human nPTB with related PTB sequences. The human nPTBsequence is aligned with PTB sequences from human (accession no.X62006), mouse (accession no. X52101),Xenopus laevis(accession no. AAF00041),D. melanogaster (accession no.AAF22979),C. elegans (accession no. CAA85411), andArabidopsis thaliana (accession no. AF076924), with apartial sequence of nPTB fromDanio rerio (accession no.AA566427) and with the sequence of human ROD1 (accession no. NM005156)using the MegAlign program (DNAStar). Residues identical to those ofnPTB are shown as dots. Brackets indicate the borders of RRM domains.(C) A phylogenetic tree of human nPTB and mammalian, plant, andC. elegans PTBs generated by MegAlign based on the proteinalignment. The length of the branch on thex axis indicatesthe percent sequence divergence.
FIG. 5
FIG. 5
Comparative analysis of the nPTB amino acid sequence.(A) Structural organization of the nPTB RRMs. RNP1 and RNP2 consensussequences for the typical RRM and for the nPTB RRMs are shown at thetop. The domain secondary structure is schematically shown below theRRM sequences. Residues identical in at least two RRMs are shaded. (B)Comparison of human nPTB with related PTB sequences. The human nPTBsequence is aligned with PTB sequences from human (accession no.X62006), mouse (accession no. X52101),Xenopus laevis(accession no. AAF00041),D. melanogaster (accession no.AAF22979),C. elegans (accession no. CAA85411), andArabidopsis thaliana (accession no. AF076924), with apartial sequence of nPTB fromDanio rerio (accession no.AA566427) and with the sequence of human ROD1 (accession no. NM005156)using the MegAlign program (DNAStar). Residues identical to those ofnPTB are shown as dots. Brackets indicate the borders of RRM domains.(C) A phylogenetic tree of human nPTB and mammalian, plant, andC. elegans PTBs generated by MegAlign based on the proteinalignment. The length of the branch on thex axis indicatesthe percent sequence divergence.
FIG. 6
FIG. 6
Tissue and cell line distribution of nPTB. (A) Northernblot analysis of poly(A)+ RNA from the indicated humantissues using an nPTB oligonucleotide probe (top) or the PTB cDNA probe(middle) probe. Loading was verified by hybridization to a β-actincontrol cDNA probe (bottom). (B) Northern blot analysis of total RNAfrom the indicated neural (lanes 1 and 2) and nonneural (lanes 3 and 4)cell lines. Probes included the full-length nPTB cDNA (top), a 1-kbfragment of PTB (middle), and the β-actin control cDNA probe(bottom). (C) Western blot analysis of proteins from neural (lanes 1 to3) and nonneural (lanes 4 to 6) cell lines using rabbit polyclonalanti-PTB antibodies recognizing both the PTB and nPTB proteins (top).The positions of the PTB isoforms and of nPTB are indicated by arrows.The same blot was probed with anti-U170K antibody as a protein-loadingcontrol (bottom).
FIG. 7
FIG. 7
Effect of PTB and nPTB on N1 exon splicing in vitro. (A)Maps of thesrc splicing substrates. Black boxes indicatesplicing-regulatory elements. (B) Splicing of adenovirus major late(lanes 1 to 8) and β-globin (lanes 9 to 16) transcripts in WERI-1nuclear extract in the absence (lanes 2 and 10) or presence of 0.3, 1,or 3 μl of HeLa (lanes 3 to 5 and 14 to 16) or WERI-1 (lanes 6 to 8and 11 to 13) 0.3 M KCl RNA affinity fractions. (C) The left panelshows splicing of the BS27 (lanes 1 to 8) or BS7 (lanes 9 to 22)transcripts in the WERI-1 nuclear extract. Lanes 1 and 9 contain noextract. Lanes 2 and 10 contain WERI extract without any additionalfactors. Lanes 3 to 5 and 14 to 16 contain WERI extract plus 0.3, 1, or3 μl of HeLa 0.3 M KCl RNA affinity fraction. Lanes 6 to 8 and 11 to13 contain WERI extract plus 0.3, 1, or 3 μl of WERI 0.3 M KCl RNAaffinity fraction. Lanes 17 to 19 contain 40, 120, or 400 ng of nPTB.Lanes 20 to 22 contain 40, 120, or 400 ng of PTB. The right panel showssplicing of BS7 transcript in the WERI-1 nuclear extract usingdifferent preparations of nPTB, PTB, and nuclear extract from the leftpanel. Lane 1 contains WERI extract without any additional factors.Lanes 2 to 5 contain 50, 100, 200, or 400 ng of nPTB. Lanes 6 to 9contain 50, 100, 200, or 400 ng of PTB.
FIG. 7
FIG. 7
Effect of PTB and nPTB on N1 exon splicing in vitro. (A)Maps of thesrc splicing substrates. Black boxes indicatesplicing-regulatory elements. (B) Splicing of adenovirus major late(lanes 1 to 8) and β-globin (lanes 9 to 16) transcripts in WERI-1nuclear extract in the absence (lanes 2 and 10) or presence of 0.3, 1,or 3 μl of HeLa (lanes 3 to 5 and 14 to 16) or WERI-1 (lanes 6 to 8and 11 to 13) 0.3 M KCl RNA affinity fractions. (C) The left panelshows splicing of the BS27 (lanes 1 to 8) or BS7 (lanes 9 to 22)transcripts in the WERI-1 nuclear extract. Lanes 1 and 9 contain noextract. Lanes 2 and 10 contain WERI extract without any additionalfactors. Lanes 3 to 5 and 14 to 16 contain WERI extract plus 0.3, 1, or3 μl of HeLa 0.3 M KCl RNA affinity fraction. Lanes 6 to 8 and 11 to13 contain WERI extract plus 0.3, 1, or 3 μl of WERI 0.3 M KCl RNAaffinity fraction. Lanes 17 to 19 contain 40, 120, or 400 ng of nPTB.Lanes 20 to 22 contain 40, 120, or 400 ng of PTB. The right panel showssplicing of BS7 transcript in the WERI-1 nuclear extract usingdifferent preparations of nPTB, PTB, and nuclear extract from the leftpanel. Lane 1 contains WERI extract without any additional factors.Lanes 2 to 5 contain 50, 100, 200, or 400 ng of nPTB. Lanes 6 to 9contain 50, 100, 200, or 400 ng of PTB.
FIG. 8
FIG. 8
Binding of purified nPTB and PTB to thesrcN1 exon splicing-regulatory elements in the presence and absence ofother protein components of the DCS complex. (A) The left panel shows agel mobility shift analysis of nPTB (lanes 2 to 6) or PTB (lanes 7 to11) complexed with an N1 3′ splice site polypyrimidine tract RNA. Lane1 contains free RNA probe (20 fmol, 105 cpm). Lanes 2 to 6contain 50, 100, 200, 400, and 800 ng of purified nPTB. Lanes 7 to 11contain equivalent amounts of PTB. The right panel shows a gel mobilityshift analysis of nPTB (lanes 2 to 6) or PTB (lanes 7 to 11) complexedwith ansrc DCS RNA. The amount of protein in each lane isequivalent to that in the left panel. (B) The left panel shows thathnRNP H enhances PTB and nPTB binding to the DCS RNA. Binding-reactionmixtures contained 100 ng of either nPTB (lanes 1 to 5) or PTB (lanes 6to 10). This was supplemented with 25 ng (lanes 1 and 6), 50 ng (lanes2 and 7), 100 ng (lanes 3 and 8), 200 ng (lanes 4 and 9), or 400 ng(lanes 5 and 10) of hnRNP H. The right panel shows assembly of theDCS-like complex from purified and recombinant factors. A total of 800ng of purified nPTB, 300 ng of recombinant hnRNP H, and/or 400 ng of aKSRP-FBP fraction were used where indicated by +. (C) DCS RNA mutantsused in the gel shift experiments. The sequence of the original DCSprobe is shown at the top. The WTs probe sequence is truncated asindicated. The ΔGTrs, ΔCUTrs, and ΔGCA mutations are indicated.Gel mobility shift analysis of nPTB complexed withsrc DCSRNA mutants in the presence of hnRNP H, KSRP, and FBP is shown belowthe sequences. A total of 200 ng of purified nPTB, 300 ng ofrecombinant hnRNP H, and/or 400 ng of a KSRP-FBP fraction were usedwhere indicated by +. The WTs and mutant probes are indicated above.The position of each complex is shown by an arrow.
FIG. 9
FIG. 9
Diagram of the DCS RNP complex. The position of eachprotein along the DCS RNA is indicated. These contacts are supported bycross-linking, affinity chromatography, and gel shift assays usingwild-type and mutant DCS RNA sequences. Homologous pairs of proteinsare indicated (hnRNPs H and F, PTB and nPTB, and KSRP and FBP). Thestoichiometry of hnRNP F and FBP binding relative to their homologs isnot yet known.
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