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

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.

FIG. 1

Structure and composition of the DCS complex. (A) nPTB cross-links to the CUCUCU element of the DCS RNA. The DCS RNA sequence is shown at the top. Modified bases are shown as hollow letters. Site-specific labeling of proteins binding to the DCS RNA is shown 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 the molecular weight standards (in thousands) are indicated on the right. (B) Immunoprecipitation of the proteins binding to the DCS RNA in the WERI 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 DCS RNA by site-specific labeling. Modified (hollow) and radiolabeled nucleotides are shown below the DCS RNA sequence. Cross-linking was done in the ASP40 fraction of WERI-1 (lanes W) or HeLa (lanes H) nuclear extract. (D) DCS RNA mutants used in the cross-linking experiments. Positions of 4-thioU modifications are indicated as U₈ and U₁₈ above the wild-type (WT) sequence, and the mutations are indicated below. (E) Effect of mutations in DCS RNA on the cross-linking of proteins to positions U₁₈ (top) and U₈ (bottom) of the DCS in the ASP40 fraction of WERI-1 extract (lanes W) or HeLa extract (lanes H).

FIG. 2

Purification of proteins bound to the DCS RNA or an unrelated RNA in the WERI-1 or HeLa extracts using RNA affinity chromatography. (A) Coomassie blue-stained gels displaying fractions from the DCS RNA affinity column (lanes 1 to 6 and lanes 8 to 13) or an unrelated RNA affinity column (lanes 15 to 20). Proteins identified by MS are indicated by arrows. Lanes 7, 14, and 21 contain protein markers. (B) Western blot analysis of the same fractions. MAb104 monoclonal antibody was used for identification of the SR proteins. The positions of the SC35 and ASF/SF2 (SRP 35), SRp55, and SRp75 proteins are indicated by arrows.

FIG. 3

Purification of human nPTB. (A) Purification scheme. Coomassie blue-stained gels of PTB- and nPTB-containing fractions from the HiTrap Blue column are shown below the scheme. (B) Coomassie blue staining of an SDS–10% polyacrylamide gel containing purified WERI-1 nPTB and HeLa PTB. (C) nPTB peptide sequences identified by Edman degradation (left) and corresponding human PTB-1 peptides (right). Differences between two proteins are shown by hollow letters.

FIG. 4

Human nPTB cDNA sequence. The NLS sequences are shaded. The four RRM domains are boxed. Sequenced peptides are in bold. The asterisk indicates the stop codon.

FIG. 5

Comparative analysis of the nPTB amino acid sequence. (A) Structural organization of the nPTB RRMs. RNP1 and RNP2 consensus sequences for the typical RRM and for the nPTB RRMs are shown at the top. The domain secondary structure is schematically shown below the RRM sequences. Residues identical in at least two RRMs are shaded. (B) Comparison of human nPTB with related PTB sequences. The human nPTB sequence 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), and Arabidopsis thaliana (accession no. AF076924), with a partial sequence of nPTB from Danio rerio (accession no. AA566427) and with the sequence of human ROD1 (accession no. NM005156) using the MegAlign program (DNAStar). Residues identical to those of nPTB are shown as dots. Brackets indicate the borders of RRM domains. (C) A phylogenetic tree of human nPTB and mammalian, plant, and C. elegans PTBs generated by MegAlign based on the protein alignment. The length of the branch on the x axis indicates the percent sequence divergence.

FIG. 5

Comparative analysis of the nPTB amino acid sequence. (A) Structural organization of the nPTB RRMs. RNP1 and RNP2 consensus sequences for the typical RRM and for the nPTB RRMs are shown at the top. The domain secondary structure is schematically shown below the RRM sequences. Residues identical in at least two RRMs are shaded. (B) Comparison of human nPTB with related PTB sequences. The human nPTB sequence 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), and Arabidopsis thaliana (accession no. AF076924), with a partial sequence of nPTB from Danio rerio (accession no. AA566427) and with the sequence of human ROD1 (accession no. NM005156) using the MegAlign program (DNAStar). Residues identical to those of nPTB are shown as dots. Brackets indicate the borders of RRM domains. (C) A phylogenetic tree of human nPTB and mammalian, plant, and C. elegans PTBs generated by MegAlign based on the protein alignment. The length of the branch on the x axis indicates the percent sequence divergence.

FIG. 6

Tissue and cell line distribution of nPTB. (A) Northern blot analysis of poly(A)⁺ RNA from the indicated human tissues using an nPTB oligonucleotide probe (top) or the PTB cDNA probe (middle) probe. Loading was verified by hybridization to a β-actin control cDNA probe (bottom). (B) Northern blot analysis of total RNA from 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-kb fragment of PTB (middle), and the β-actin control cDNA probe (bottom). (C) Western blot analysis of proteins from neural (lanes 1 to 3) and nonneural (lanes 4 to 6) cell lines using rabbit polyclonal anti-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-loading control (bottom).

FIG. 7

Effect of PTB and nPTB on N1 exon splicing in vitro. (A) Maps of the src splicing substrates. Black boxes indicate splicing-regulatory elements. (B) Splicing of adenovirus major late (lanes 1 to 8) and β-globin (lanes 9 to 16) transcripts in WERI-1 nuclear 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 8 and 11 to 13) 0.3 M KCl RNA affinity fractions. (C) The left panel shows 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 no extract. Lanes 2 and 10 contain WERI extract without any additional factors. Lanes 3 to 5 and 14 to 16 contain WERI extract plus 0.3, 1, or 3 μl of HeLa 0.3 M KCl RNA affinity fraction. Lanes 6 to 8 and 11 to 13 contain WERI extract plus 0.3, 1, or 3 μl of WERI 0.3 M KCl RNA affinity 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 shows splicing of BS7 transcript in the WERI-1 nuclear extract using different preparations of nPTB, PTB, and nuclear extract from the left panel. 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 9 contain 50, 100, 200, or 400 ng of PTB.

FIG. 7

Effect of PTB and nPTB on N1 exon splicing in vitro. (A) Maps of the src splicing substrates. Black boxes indicate splicing-regulatory elements. (B) Splicing of adenovirus major late (lanes 1 to 8) and β-globin (lanes 9 to 16) transcripts in WERI-1 nuclear 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 8 and 11 to 13) 0.3 M KCl RNA affinity fractions. (C) The left panel shows 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 no extract. Lanes 2 and 10 contain WERI extract without any additional factors. Lanes 3 to 5 and 14 to 16 contain WERI extract plus 0.3, 1, or 3 μl of HeLa 0.3 M KCl RNA affinity fraction. Lanes 6 to 8 and 11 to 13 contain WERI extract plus 0.3, 1, or 3 μl of WERI 0.3 M KCl RNA affinity 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 shows splicing of BS7 transcript in the WERI-1 nuclear extract using different preparations of nPTB, PTB, and nuclear extract from the left panel. 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 9 contain 50, 100, 200, or 400 ng of PTB.

FIG. 8

Binding of purified nPTB and PTB to the src N1 exon splicing-regulatory elements in the presence and absence of other protein components of the DCS complex. (A) The left panel shows a gel mobility shift analysis of nPTB (lanes 2 to 6) or PTB (lanes 7 to 11) complexed with an N1 3′ splice site polypyrimidine tract RNA. Lane 1 contains free RNA probe (20 fmol, 10⁵ cpm). Lanes 2 to 6 contain 50, 100, 200, 400, and 800 ng of purified nPTB. Lanes 7 to 11 contain equivalent amounts of PTB. The right panel shows a gel mobility shift analysis of nPTB (lanes 2 to 6) or PTB (lanes 7 to 11) complexed with an src DCS RNA. The amount of protein in each lane is equivalent to that in the left panel. (B) The left panel shows that hnRNP H enhances PTB and nPTB binding to the DCS RNA. Binding-reaction mixtures contained 100 ng of either nPTB (lanes 1 to 5) or PTB (lanes 6 to 10). This was supplemented with 25 ng (lanes 1 and 6), 50 ng (lanes 2 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 the DCS-like complex from purified and recombinant factors. A total of 800 ng of purified nPTB, 300 ng of recombinant hnRNP H, and/or 400 ng of a KSRP-FBP fraction were used where indicated by +. (C) DCS RNA mutants used in the gel shift experiments. The sequence of the original DCS probe is shown at the top. The WTs probe sequence is truncated as indicated. The ΔGTrs, ΔCUTrs, and ΔGCA mutations are indicated. Gel mobility shift analysis of nPTB complexed with src DCS RNA mutants in the presence of hnRNP H, KSRP, and FBP is shown below the sequences. A total of 200 ng of purified nPTB, 300 ng of recombinant hnRNP H, and/or 400 ng of a KSRP-FBP fraction were used where indicated by +. The WTs and mutant probes are indicated above. The position of each complex is shown by an arrow.

FIG. 9

Diagram of the DCS RNP complex. The position of each protein along the DCS RNA is indicated. These contacts are supported by cross-linking, affinity chromatography, and gel shift assays using wild-type and mutant DCS RNA sequences. Homologous pairs of proteins are indicated (hnRNPs H and F, PTB and nPTB, and KSRP and FBP). The stoichiometry of hnRNP F and FBP binding relative to their homologs is not yet known.