The Polypyrimidine Tract Binding Protein (PTB) Represses Splicing of Exon 6B from the β-Tropomyosin Pre-mRNA by Directly Interfering with the Binding of the U2AF65 Subunit

Abstract

Splicing of exon 6B from the ␤-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.Alternative splicing is a widespread mechanism that increases protein diversity and regulates gene expression in higher eukaryotes. This process is particularly prominent in humans, as it has been estimated that at least 60% of the human genes are alternatively spliced. Alternative splicing generates several mRNAs from a single gene, leading to the synthesis of several proteins with distinct biological functions, different intracellular localizations, or different stabilities (reviewed in reference 47). Thus, alternative splicing plays a major role in defining the repertoire of proteins that are expressed in different cells. From numerous studies, it appears that the regulation of alternative splicing results from a complex interplay between multiple trans-acting factors and cis-acting sequences that facilitates or prevents the recruitment of splicing factors by the splicing machinery (reviewed in references 7 and 44).The polypyrimidine tract binding protein (PTB), also known as hnRNP I, is one of the best-characterized splicing repressors. As demonstrated recently, PTB is an essential protein, needed for the development of Xenopus laevis embryos (28). Consistent with its widespread expression, PTB has been implicated in the repression of a large number of alternative splicing events (reviewed in references 7, 48, and 51). PTB recognizes short motifs, such as UCUU and UCUCU, located within a pyrimidine-rich context and often associated with the polypyrimidine tract upstream of the 3Ј splice site of alternative exons (3,8,9,21,37). However, binding sites for PTB have also been found in exonic sequences and in introns downstream of regulated exons (13,23,27,40). In most alternative splicing systems regulated by PTB, repression is achieved through the interaction of PTB with multiple PTB binding sites surrounding the alternative exon (3, 9-11...

Structure-function relationships of the polypyrimidine tract binding protein

“…65,2008 Review Article 521 to function through an interaction with PTB, and might facilitate the identification of novel PTB targets. For example, many studies identified PTB binding sites by boundary analyses, gel shift experiments and cross-linking [10,15,22,32]. In light of the complex structures [85], these findings can be more easily interpreted.…”

“…The distribution of PTB binding sites within introns has led to several proposed models of PTB action. For example, PTB could interfere with spliceosome assembly by binding to the branch point pyrimidine tract and therefore directly sequestering the branch-point or competing with U2AF, an essential component of the constitutive splicing machinery [2,15,22,32]. In most cases, however, the exons silenced by PTB are flanked by PTB binding sites on both adjacent introns, and mutations of upstream PTB binding sequences have been shown to reduce binding of PTB to the downstream sites [7].…”

“…While all three RRMs are likely to be accommodated on the 20-nucleotide site, only two RRMs could bind on the remaining RNA, which will lower the affinity for the second molecule. Furthermore, in splicing regulation, it has been previously proposed [2] and more recently shown [15,32] that PTB might function by displacing U2AF, a factor essential for the initiation of a successful splicing event, from the branch point pyrimidine tract. U2AF has been shown to bind specifically to poly(U) tracts [2,89].…”

Polypyrimidine Tract Binding Protein Homologs from Arabidopsis Are Key Regulators of Alternative Splicing with Implications in Fundamental Developmental Processes

“…One example for positiondependent splicing is constituted by the hnRNP protein PTB (Xue et al, 2009;Llorian et al, 2010), an in animals, wellcharacterized regulator of AS that binds to pyrimidine-rich motifs within pre-mRNAs (Sawicka et al, 2008;Wachter et al, 2012). Evidence has been provided that PTB exploits various mechanisms for AS control, including competing with U2 auxiliary factor 65 in binding to the pre-mRNA (Saulière et al, 2006), looping of RNA regions (Spellman and Smith, 2006), and interference with splicing factor interactions required for exon or intron definition (Izquierdo et al, 2005;Sharma et al, 2005). In animals, a switch in expression from PTB to its neuronal homolog nPTB was shown to reprogram AS patterns and coincides with neuronal development (Boutz et al, 2007).…”

Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3′-Terminal Exon

“…The observation that in the absence of UTE, a 2-fold increase in exon 9A9Ј usage was still observed when PTB was down-regulated strongly suggests that in addition to precluding the assembly of an activator complex onto UTE, PTB directly inhibits the 3Ј splice site and ␣e poly(A) site selection by the basal splicing and cleavage/polyadenylation machineries. Since numerous high affinity PTB binding sites are present between the Ϫ274 distal branch site and exon 9A9Ј, it is likely that PTB inhibits the splicing of exon 9A9Ј by directly preventing the binding of U2AF65, as described for the mutually exclusive exon 6B of the ␤-tropomyosin pre-mRNA (21). It is also conceivable that PTB blocks the transition from an exon 9A9Ј defined complex to a functional spliceosome, as demonstrated for the regulation of the alternative exon N1 of the c-src pre-mRNA (26).…”