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.Alternative splicing is a common mechanism for regulating gene expression in eukaryotes, allowing the generation of diverse proteins from the same primary RNA transcript (46,77,78). The alteration of splice site choice is thought to be determined by regulatory proteins that bind to the pre-mRNA transcript and affect spliceosome assembly on particular exons or splice sites. The best characterized of these splicing-regulatory proteins are a set of polypeptides called SR proteins that, among many other properties, bind to exonic splicing enhancer sequences (7,10,35,47,75). The SR proteins bound to an exonic enhancer are thought to stimulate spliceosome assembly at the adjacent splice sites. Another group of pre-mRNA binding proteins are the heterogeneous nuclear ribonucleoproteins (hnRNPs) (19,66). These are a diverse group of molecules that coat nascent pre-mRNAs, forming complex but little understood hnRNP structures (42, 52). The assembly of the spliceosome occurs after formation of these hnRNP complexes, and some hnRNPs have been implicated in splicing regulation. For example, hnRNP A1 is able to counteract the effect of SR proteins in some assays and can also apparently repress splicing through splicing silencer sequences (3,7,8,11,31). However, the assembly of a pre-mRNP complex is poorly understood. It is apparently highly cooperative, but the interactions between the different hnRNPs in these complexes are mostly unknown.Although widely expressed, the SR proteins and hnRNPs do vary in concentration between different tissues (31, 39). Changes in splicing patterns are thought to be determined, in part, ...

We have purified and cloned a new splicing factor, KSRP. KSRP is a component of a multiprotein complex that binds specifically to an intronic splicing enhancer element downstream of the neuron-specific c-src N1 exon. This 75-kD protein induces the assembly of five other proteins, including the heterogeneous nuclear ribonucleoprotein F, onto the splicing enhancer. The sequence of the KSRP cDNA indicates that the protein contains four K homology RNA-binding domains and an unusual carboxy-terminal domain. KSRP is similar to two proteins, FUSE-binding protein and P-element somatic inhibitor. KSRP is expressed in both neural and non-neural cell lines, although it is present at higher levels in neural cells. Antibodies specific for KSRP inhibit the splicing of the N1 exon in vitro. Moreover, this inhibition of N1 splicing can be rescued by the addition of purified KSRP. KSRP is likely to regulate splicing from a number of intronic splicing enhancer sequences.[Key Words: Alternative splicing; regulatory protein; KSRP; intronic splicing enhancer; RNA-binding protein]

We studied the role of polypyrimidine tract binding protein in repressing splicing of the c-src neuron-specific N1 exon. Immunodepletion/add-back experiments demonstrate that PTB is essential for splicing repression in HeLa extract. When splicing is repressed, PTB cross-links to intronic CUCUCU elements flanking the N1 exon. Mutation of the downstream CU elements causes dissociation of PTB from the intact upstream CU elements and allows splicing. Thus, PTB molecules bound to multiple elements cooperate to repress splicing. Interestingly, in neuronal WERI-1 cell extract where N1 is spliced, PTB also binds to the upstream CU elements but is dissociated in the presence of ATP. We conclude that splicing repression by PTB is modulated in different cells by a combination of cooperative binding and ATP-dependent dissociation.