SC35 belongs to the family of SR proteins that regulate alternative splicing in a concentration-dependent manner in vitro and in vivo. We previously reported that SC35 is expressed through alternatively spliced mRNAs with differing 3¢ untranslated sequences and stabilities. Here, we show that overexpression of SC35 in HeLa cells results in a signi®cant decrease of endogenous SC35 mRNA levels along with changes in the relative abundance of SC35 alternatively spliced mRNAs. Remarkably, SC35 leads to both an exon inclusion and an intron excision in the 3¢ untranslated region of its mRNAs. In vitro splicing experiments performed with recombinant SR proteins demonstrate that SC35, but not ASF/SF2 or 9G8, speci®cally activates these alternative splicing events. Interestingly, the resulting mRNA is very unstable and we present evidence that mRNA surveillance is likely to be involved in this instability. SC35 therefore constitutes the ®rst example of a splicing factor that controls its own expression through activation of splicing events leading to expression of unstable mRNA. Keywords: alternative splicing/autoregulation/mRNA stability/SC35 IntroductionAlternative splicing, allowing the formation of different mRNA isoforms from a single gene, is a fundamental process controlling genetic expression in higher eukaryotes. Among the different factors involved in the regulation of alternative splicing, one group of non-snRNP proteins belongs to a conserved family of structurally and functionally related phosphoproteins called SR proteins (for review see Fu, 1995;Manley and Tacke, 1996;Graveley, 2000). SR proteins exhibit a modular structure consisting of one or two RNA recognition motifs (RRMs) and a C-terminal domain of variable length (the RS domain), rich in Arg and Ser residues. Whereas the RS domain is responsible for speci®c protein±protein interactions, the RRM domain binds to many different sites along the RNA and is involved in speci®c interactions with sequences, known as exonic (ESE) or intronic splicing enhancers (ISE), identi®ed in numerous pre-mRNAs (for reviews see Tacke and Manley, 1999;Graveley, 2000).Several studies have reported that SR proteins, which are involved in the early steps of spliceosome assembly, promote binding of the U1 snRNP to the 5¢ splice site and stimulate binding of U2AF and the U2 snRNP to the 3¢ splice site region (Reed, 1996 and references therein). Since the RS domains of SR factors interact with themselves and with each other, as well as with the U1-70K and U2AF 35 proteins, it has been proposed that a role of SR proteins consists of bringing the 5¢ and 3¢ splice sites together via a bridge of protein contacts (for review see Graveley, 2000). In addition to their role in constitutive splicing, SR proteins also in¯uence the selection of alternative splice sites in a concentrationdependent manner both in vitro and in transfected cells (for review see Smith and Valcarcel, 2000). For some, if not all SR proteins, this activity can be antagonized by members of the hnRNP A/B family (Fu ...

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...