of splice site selection. Clearly other elements, in cis and Alain Expert-Bezanç on in trans, are needed to define which splice site will Centre de Génétique Moléculaire, Centre National de la Recherche be chosen by the splicing machinery in a given cell Scientifique, 91190 (Mardon et al., 1987;Hampson et al., 1989;Streuli and Saito, 1989; Helfman Exons 6A and 6B of the chicken β-tropomyosin gene et al., 1990;Libri et al., 1990;Black, 1992; Lavigueur are mutually exclusive and selected in a tissue- specific et al., 1993;Sun et al., 1993;Watakabe et al., 1993; manner. Exon 6A is present in non-muscle and smooth Gooding et al., 1994;Del Gatto and Breathnach, 1995; muscle cells, while exon 6B is present in skeletal muscle Ryan and Cooper, 1996). Notable among these, purinecells. In this study we have investigated the mechanism rich elements identified in several vertebrate exons, called underlying exon 6A recognition in non-muscle cells.splicing enhancers, are required for efficient splicing of Previous reports have identified a pyrimidine-rich the resident exons. In general, exonic splicing enhancers intronic enhancer sequence (S4) downstream of exon appear to activate splicing of the upstream intron 6A as essential for exon 6A 5Ј-splice site recognition. (Lavigueur et al., 1993;Watakabe et al., 1993; Xu We show here that preincubation of HeLa cell extracts et al., 1993). These enhancer sequences contain contiguous with an excess of RNA containing this sequence sperepeats of the motif GARGAR (R ϭ purine). Specific cifically inhibits exon 6A recognition by the splicing binding of SR proteins has been shown for enhancers machinery. Splicing inhibition by an excess of this present in exons of several genes. A direct correlation RNA can be rescued by addition of the SR protein between SR protein binding and activation of splicing has ASF/SF2, but not by the SR proteins SC35 or 9G8.been established for ASF/SF2 in the case of the growth ASF/SF2 stimulates exon 6A splicing through specific hormone gene (Sun et al., 1993) and SRp40 and SRp55 interaction with the enhancer sequence. Surprisingly, in the case of the troponin T gene (Ramchatesingh et al., SC35 behaves as an inhibitor of exon 6A splicing, 1995). The SR proteins are essential splicing factors since addition to HeLa nuclear extracts of increasing implicated in early steps of the spliceosome formation amounts of the SC35 protein completely abolish the pathway (Krainer et al., 1990;Fu and Maniatis, 1992b). stimulatory effect of ASF/SF2 on exon 6A splicing. WeIn fact, commitment of a pre-mRNA to splicing requires conclude that exon 6A recognition in vitro depends on binding of a specific SR protein to the pre-mRNA (Fu, the ratio of the ASF/SF2 to SC35 SR proteins. Taken 1993;Kohtz et al., 1994;Staknis and Reed, 1994). Studies together our results suggest that variations in the level of in vitro protein-protein interactions and using the yeast or activity of these proteins could contribute to the two-hybrid system show that the individual RS domains tissue-sp...

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...

Mutually exclusive splicing of exons 6A and 6B from the chicken ␤-tropomyosin gene involves numerous regulatory sequences. Previously, we identified a G-rich intronic sequence (S3) downstream of exon 6B. This element consists of six G-rich motifs, mutations of which abolish splicing of exon 6B. In this paper, we investigated the cellular factors that bind to this G-rich element. By using RNA affinity chromatography, we identified heterogeneous nuclear ribonucleoprotein (hnRNP) A1, the SR proteins ASF/SF2 and SC35, and hnRNP F/H as specific components that are assembled onto the G-rich element. By using hnRNP A1-depleted HeLa nuclear extract and add-back experiments, we show that hnRNP A1 has a negative effect on splicing of exon 6B. In agreement with in vitro data, artificial recruitment of hnRNP A1, as a fusion with the MS2 coat protein, also represses splicing of exon 6B ex vivo. In contrast, ASF/SF2 and SC35 activate splicing of exon 6B. As observed with other systems, hnRNP A1 counteracts the stimulating effect of the SR proteins. Moreover, cross-linking experiments show that both ASF/SF2 and SC35 are able to displace binding of hnRNP A1 to the G-rich element, suggesting that the binding sites for these proteins are overlapping. These data indicate that the G-rich sequence is a composite element that acts as an enhancer or as a silencer, depending on which proteins bind to them. Splicing is the process by which introns from premessenger RNAs are removed in eukaryotes. Pre-mRNA splicing takes place within the spliceosome, which is a large, highly dynamic complex composed of four small ribonucleoprotein particles (snRNP 1 U1, U2, U4/U6, and U5) and multiple non-snRNP factors (1, 2). One of the most intriguing questions that remains in RNA splicing is how the 5Ј and 3Ј splice sites are selected and paired together within large RNA sequences (3). This question takes on particular importance in alternative splicing, where the selection of certain splice sites is modulated depending on the developmental stage, on tissue differentiation, or on metabolic changes of the cells (3). Numerous studies have demonstrated that regulatory sequences within the pre-mRNA that lie outside the splicing signals play a crucial role in controlling the choice of splicing sites in a given cellular context (reviewed in Refs. 4 and 5).Among these sequences are the splicing enhancers. These elements are found in a wide variety of metazoan pre-mRNAs, either within exons or introns. Purine-rich splicing enhancers (known as ESE) are a well characterized class of exonic splicing enhancers that mostly interact with specific subsets of SR proteins (reviewed in Refs. 6 and 7). SR proteins belong to a family of essential splicing factors that are highly conserved between Drosophila and mammals and that are involved in both constitutive and regulated splicing events (reviewed in Refs. 8 and 9). It has been proposed that the function of SR proteins is to stimulate the recognition of weak upstream 3Ј splice sites, by recruiting U2AF 65/35 , or to f...