Exon repression by polypyrimidine tract binding protein

Polypyrimidine tract binding protein (PTB) is known to silence the splicing of many alternative exons. However, exons repressed by PTB are affected by other RNA regulatory elements and proteins. This makes it difficult to dissect the structure of the pre-mRNP complexes that silence splicing, and to understand the role of PTB in this process. We determined the minimal requirements for PTB-mediated splicing repression. We find that the minimal sequence for high affinity binding by PTB is relatively large, containing multiple polypyrimidine elements. Analytical ultracentrifugation and proteolysis mapping of RNA cross-links on the PTB protein indicate that most PTB exists as a monomer, and that a polypyrimidine element extends across multiple PTB domains. The high affinity site is bound initially by a PTB monomer and at higher concentrations by additional PTB molecules. Significantly, this site is not sufficient for splicing repression when placed in the 3' splice site of a strong test exon. Efficient repression requires a second binding site within the exon itself or downstream from it. This second site enhances formation of a multimeric PTB complex, even if it does not bind well to PTB on its own. These experiments show that PTB can be sufficient to repress splicing of an otherwise constitutive exon, without binding sites for additional regulatory proteins and without competing with U2AF binding. The minimal complex mediating splicing repression by PTB requires two binding sites bound by an oligomeric PTB complex.     

The polypyrimidine tract binding protein (PTB) represses splicing of exon 6B from the beta-tropomyosin pre-mRNA by directly interfering with the binding of the U2AF65 subunit

Splicing of exon 6B from the beta-tropomyosin pre-mRNA is repressed in nonmuscle cells and myoblasts by a complex array of intronic elements surrounding the exon. In this study, we analyzed the proteins that mediate splicing repression of exon 6B through binding to the upstream element. We identified the polypyrimidine tract binding protein (PTB) as a component of complexes isolated from myoblasts that assemble onto the branch point region and the pyrimidine tract. In vitro splicing assays and PTB knockdown experiments by RNA interference demonstrated that PTB acts as a repressor of splicing of exon 6B. Using psoralen experiments, we showed that PTB acts at an early stage of spliceosome assembly by preventing the binding of U2 snRNA on the branch point. Using UV cross-linking and immunoprecipitation experiments with site-specific labeled RNA in PTB-depleted nuclear extracts, we found that the decrease in PTB was correlated with an increase in U2AF65. In addition, competition experiments showed that PTB is able to displace the binding of U2AF65 on the polypyrimidine tract. Our results strongly support a model whereby PTB competes with U2AF65 for binding to the polypyrimidine tract.     

A splicing repressor domain in polypyrimidine tract-binding protein

Polypyrimidine tract-binding protein (PTB) is an hnRNP with four RRM type domains. It plays roles as a repressive alternative splicing regulator of multilple target genes, as well as being involved in pre-mRNA 3' end processing, mRNA localization, stability, and internal ribosome entry site-mediated translation. Here we have used a tethered function assay, in which a fusion protein of PTB and the bacteriophage MS2 coat protein is recruited to a splicing regulatory site by binding to an artificially inserted MS2 binding site. Deletion mutations of PTB in this system allowed us to identify RRM2 and the following inter-RRM linker region as the minimal region of PTB that can act as splicing repressor domain when recruited to RNA. Splicing repression by the minimal repressor domain remained cell type-specific and dependent upon other defined regulatory elements in the alpha-tropomyosin test minigene. Our results highlight the fact that splicing repression by PTB can be uncoupled from the mode by which it binds to RNA.     

The polypyrimidine tract binding protein binds upstream of neural cell-specific c-src exon N1 to repress the splicing of the intron downstream.

The neural cell-specific N1 exon of the c-src pre-mRNA is both negatively regulated in nonneural cells and positively regulated in neurons. We previously identified conserved intronic elements flanking N1 that direct the repression of N1 splicing in a nonneural HeLa cell extract. The upstream repressor elements are located within the polypyrimidine tract of the N1 exon 3' splice site. A short RNA containing this 3' splice site sequence can sequester trans-acting factors in the HeLa extract to allow splicing of N1. We now show that these upstream repressor elements specifically interact with the polypyrimidine tract binding protein (PTB). Mutations in the polypyrimidine tract reduce both PTB binding and the ability of the competitor RNA to derepress splicing. Moreover, purified PTB protein restores the repression of N1 splicing in an extract derepressed by a competitor RNA. In this system, the PTB protein is acting across the N1 exon to regulate the splicing of N1 to the downstream exon 4. This mechanism is in contrast to other cases of splicing regulation by PTB, in which the protein represses the splice site to which it binds.     

Neuronal regulation of pre-mRNA splicing by polypyrimidine tract binding proteins,PTBP1 and PTBP2

Alternative splicing patterns are regulated by RNA binding proteins that assemble onto each pre-mRNA to form a complex RNP structure. The polypyrimidine tract binding protein, PTB, has served as an informative model for understanding how RNA binding proteins affect spliceosome assembly and how changes in the expression of these proteins can control complex programs of splicing in tissues. In this review, we describe the mechanisms of splicing regulation by PTB and its function, along with its paralog PTBP2, in neuronal development.     

Neuronal regulation of pre-mRNA splicing by polypyrimidine tract binding proteins, PTBP1 and PTBP2

Alternative splicing patterns are regulated by RNA binding proteins that assemble onto each pre-mRNA to form a complex RNP structure. The polypyrimidine tract binding protein, PTB, has served as an informative model for understanding how RNA binding proteins affect spliceosome assembly and how changes in the expression of these proteins can control complex programs of splicing in tissues. In this review, we describe the mechanisms of splicing regulation by PTB and its function, along with its paralog PTBP2, in neuronal development.     

Differential alternative splicing activity of isoforms of polypyrimidine tract binding protein (PTB).

Polypyrimidine tract binding protein (PTB) is an RNA-binding protein that regulates splicing by repressing specific splicing events. It also has roles in 3'-end processing, internal initiation of translation, and RNA localization. PTB exists in three alternatively spliced isoforms, PTB1, PTB2, and PTB4, which differ by the insertion of 19 or 26 amino acids, respectively, between the second and third RNA recognition motif domains. Here we show that the PTB isoforms have distinct activities upon alpha-tropomyosin (TM) alternative splicing. PTB1 reduced the repression of TM exon 3 in transfected smooth muscle cells, whereas PTB4 enhanced TM exon 3 skipping in vivo and in vitro. PTB2 had an intermediate effect. The PTB4 > PTB2 > PTB1 repressive hierarchy was observed in all in vivo and in vitro assays with TM, but the isoforms were equally active in inducing skipping of alpha-actinin exons and showed the opposite hierarchy of activity when tested for activation of IRES-driven translation. These findings establish that the ratio of PTB isoforms could form part of a cellular code that in turn controls the splicing of various other pre-mRNAs.     

Structure and RNA interactions of the N-terminal RRM domains of PTB

The polypyrimidine tract binding protein (PTB) is an important regulator of alternative splicing that also affects mRNA localization, stabilization, polyadenylation, and translation. NMR structural analysis of the N-terminal half of PTB (residues 55-301) shows a canonical structure for RRM1 but reveals novel extensions to the beta strands and C terminus of RRM2 that significantly modify the beta sheet RNA binding surface. Although PTB contains four RNA recognition motifs (RRMs), it is widely held that only RRMs 3 and 4 are involved in RNA binding and that RRM2 mediates homodimerization. However, we show here not only that the RRMs 1 and 2 contribute substantially to RNA binding but also that full-length PTB is monomeric, with an elongated structure determined by X-ray solution scattering that is consistent with a linear arrangement of the constituent RRMs. These new insights into the structure and RNA binding properties of PTB suggest revised models of its mechanism of action.     

Mutations in RRM4 uncouple the splicing repression and RNA-binding activities of polypyrimidine tract binding protein

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequence-specific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays.     

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

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.     

Crossregulation and functional redundancy between the splicing regulator PTB and its paralogs nPTB and ROD1

Among the targets of the repressive splicing regulator, polypyrimidine tract binding protein (PTB) is its own pre-mRNA, where PTB-induced exon 11 skipping produces an RNA substrate for nonsense-mediated decay (NMD). To identify additional PTB-regulated alternative splicing events, we used quantitative proteomic analysis of HeLa cells after knockdown of PTB. Apart from loss of PTB, the only change was upregulation of the neuronally restricted nPTB, resulting from decreased skipping of nPTB exon 10, a splicing event that leads to NMD of nPTB mRNA. Compared with knockdown of PTB alone, simultaneous knockdown of PTB and nPTB led to larger changes in alternative splicing of known and newly identified PTB-regulated splicing events. Strikingly, the hematopoietic PTB paralog ROD1 also switched from a nonproductive splicing pathway upon PTB/nPTB knockdown. Our data indicate crossregulation between PTB and its paralogs via nonproductive alternative splicing and a large degree of functional overlap between PTB and nPTB.     

Coordinate repression of a trio of neuron-specific splicing events by the splicing regulator PTB.

In this study, we demonstrate the ability of the polypyrimidine tract binding protein PTB to function as a coordinator of splicing regulation for a trio of neuron-specific exons that are subject to developmental splicing changes in the rat cerebellum. Three neuron-specific exons that show positive regulation are derived from the GABA(A) receptor gamma2 subunit 24 nucleotide exon, clathrin light chain B exon EN, and N-methyl-D-aspartate receptor NR1 subunit exon 5 pre-mRNAs. The functional activity of splicing repressor signals located in the 3' splice site regions adjacent to the neural exons is shown using an alternative splicing switch assay, in which these short RNA sequences function in trans to switch splicing to the neural pathway in HeLa splicing reactions. Parallel UV crosslinking/competition assays demonstrate selective binding of PTB in comparison to substantially lower binding at adjacent, nonneural 3' splice sites. Substantially lower PTB binding and splicing switch activity is also observed for the 3' splice site of NMDA exon 21, which is subject to negative regulation in cerebellum tissue in the same time frame. In splicing active neural extracts, the balance of control shifts to positive regulation, and this shift correlates with a PTB status that is predominantly the neural form. In this context, the addition of recombinant PTB is sufficient to switch splicing to the nonneural pathway. The neural extracts also reveal specific binding of the CUG triplet repeat binding protein to a subset of regulatory 3' splice site regions. These interactions may interfere with PTB function or modulate splicing levels in a substrate-specific manner within neural tissue. Together these results strengthen the evidence that PTB is a splicing regulator with multiple targets and demonstrate its ability to discriminate among neural and nonneural substrates. Thus, a variety of mechanisms that counterbalance the splicing repressor function of PTB in neural tissue are capable of mediating developmental splicing control. Altered expression of PTB isoforms during cerebellar development, as documented by Western blot analysis, is proposed to be a contributing mechanism.     

Structure of tandem RNA recognition motifs from polypyrimidine tract binding protein reveals novel features of the RRM fold

Polypyrimidine tract binding protein (PTB), an RNA binding protein containing four RNA recognition motifs (RRMs), is involved in both pre-mRNA splicing and translation initiation directed by picornaviral internal ribosome entry sites. Sequence comparisons previously indicated that PTB is a non-canonical RRM protein. The solution structure of a PTB fragment containing RRMs 3 and 4 shows that the protein consists of two domains connected by a long, flexible linker. The two domains tumble independently in solution, having no fixed relative orientation. In addition to the betaalphabetabetaalphabeta topology, which is characteristic of RRM domains, the C-terminal extension of PTB RRM-3 incorporates an unanticipated fifth beta-strand, which extends the RNA binding surface. The long, disordered polypeptide connecting beta4 and beta5 in RRM-3 is poised above the RNA binding surface and is likely to contribute to RNA recognition. Mutational analyses show that both RRM-3 and RRM-4 contribute to RNA binding specificity and that, despite its unusual sequence, PTB binds RNA in a manner akin to that of other RRM proteins.     

PTB蛋白:一种调控肿瘤代谢的RNA可变剪接调节因子

多聚嘧啶区结合蛋白(polypyrimidine tract binding protein,PTB或hnRNP I)是一种在细胞内部参与mRNA代谢过程的蛋白质。PTB蛋白可结合于核酸分子上富含嘧啶碱基的序列,对mRNA前体的剪接进行调控。如在部分肿瘤细胞中,PTB的表达量升高可对肿瘤代谢过程中关键的丙酮酸激酶M(pyruvate kinase M,PKM)基因的表达进行调控,通过抑制PKM基因的可变剪接的方式上调PKM2(pyruvate kinase M2,PKM2)的表达,进而强化肿瘤细胞的有氧糖酵解过程并促进肿瘤的发展。本文结合PTB蛋白的结构及其在PKM可变剪接过程中的调节机制,简要综述了PTB蛋白对肿瘤代谢的调控作用。     

Crystallographic analysis of polypyrimidine tract-binding protein-Raver1 interactions involved in regulation of alternative splicing

The polypyrimidine tract-binding protein (PTB) is an important regulator of alternative splicing. PTB-regulated splicing of α-tropomyosin is enhanced by Raver1, a protein with four PTB-Raver1 interacting motifs (PRIs) that bind to the helical face of the second RNA recognition motif (RRM2) in PTB. We present the crystal structures of RRM2 in complex with PRI3 and PRI4 from Raver1, which--along with structure-based mutagenesis--reveal the molecular basis of their differential binding. High-affinity binding by Raver1 PRI3 involves shape-matched apolar contacts complemented by specific hydrogen bonds, a new variant of an established mode of peptide-RRM interaction. Our results refine the sequence of the PRI motif and place important structural constraints on functional models of PTB-Raver1 interactions. Our analysis indicates that the observed Raver1-PTB interaction is a general mode of binding that applies to Raver1 complexes with PTB paralogues such as nPTB and to complexes of Raver2 with PTB.     

Mutation of PTB binding sites causes misregulation of alternative 3' splice site selection in vivo.

Alternative splicing of pre-mRNA is a commonly used mechanism to regulate gene expression in higher eukaryotes. However, with the exception of regulated cascades in Drosophila, the cis-acting elements and the trans-acting factors that control tissue- and/or developmentally regulated splicing remain largely unidentified. Cis-acting elements that control smooth muscle-specific repression of exon 3 of alpha-tropomyosin (alpha-TM) have been identified recently and consist of two regions that flank this exon. Deletion of either element causes misregulated splicing of alpha-TM in transfected smooth muscle cells. In experiments designed to characterize essential sequences within each element and the factors that interact with these sequences, we have identified two overlapping sequences within the downstream regulatory element (DRE) that are identical to binding sites for polypyrimidine tract binding protein (PTB) that were identified using iterative selection techniques. Mutation of these sites caused aberrant splicing regulation in transfected smooth muscle cells. In addition, sequences identical to high-affinity PTB binding sites were also detected upstream of exon 3 and mutation of these sites also resulted in misregulation of splicing in vivo, suggesting that PTB binding to specific sequences flanking exon 3 is responsible, in part, for the repression of exon 3. Consistent with this hypothesis, UV crosslinking and equilibrium binding assays confirm that the same mutations that cause misregulated splicing also disrupt PTB binding to RNA.     

Polypyrimidine tract binding protein modulates efficiency of polyadenylation

Polypyrimidine tract binding protein (PTB) is a major hnRNP protein with multiple roles in mRNA metabolism, including regulation of alternative splicing and internal ribosome entry site-driven translation. We show here that a fourfold overexpression of PTB results in a 75% reduction of mRNA levels produced from transfected gene constructs with different polyadenylation signals (pA signals). This effect is due to the reduced efficiency of mRNA 3' end cleavage, and in vitro analysis reveals that PTB competes with CstF for recognition of the pA signal's pyrimidine-rich downstream sequence element. This may be analogous to its role in alternative splicing, where PTB competes with U2AF for binding to pyrimidine-rich intronic sequences. The pA signal of the C2 complement gene unusually possesses a PTB-dependent upstream sequence, so that knockdown of PTB expression by RNA interference reduces C2 mRNA expression even though PTB overexpression still inhibits polyadenylation. Consequently, we show that PTB can act as a regulator of mRNA expression through both its negative and positive effects on mRNA 3' end processing.     

Conformation of polypyrimidine tract binding protein in solution

The polypyrimidine tract binding protein (PTB) is an RNA binding protein that normally functions as a regulator of alternative splicing but can also be recruited to stimulate translation initiation by certain picornaviruses. High-resolution structures of the four RNA recognition motifs (RRMs) that make up PTB have previously been determined by NMR. Here, we have used small-angle X-ray scattering to determine the low-resolution structure of the entire protein. Scattering patterns from full-length PTB and deletion mutants containing all possible sequential combinations of the RRMs were collected. All constructs were found to be monomeric in solution. Ab initio analysis and rigid-body modeling utilizing the high-resolution models of the RRMs yielded a consistent low-resolution model of the spatial organization of domains in PTB. Domains 3 and 4 were found to be in close contact, whereas domains 2 and especially 1 had loose contacts with the rest of the protein.