Cooperative Assembly of an hnRNP Complex Induced by a Tissue-Specific Homolog of Polypyrimidine Tract Binding Protein

Abstract

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, ...

Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors

“…There is additionally a version of the protein termed nPTB that shows enhanced expression in neuronal tissue. 39 PTB contains four loosely conserved RNA recognition motifs (RRMs) 40 with the isoform-specific insertions being between RRMs 2 and 3, near to a linker, or hinge, region. The RRMs of PTB contain unexpected amino acids compared with the RRM family consensus, particularly in their lack of conserved aromatic residues, these being important in other RRMs for nonspecific RNA contact; most of all at position 2 of the second part of the RRM (RNP-1) where a conserved glycine is absent in all four PTB RRMs.…”

Structure-function relationships of the polypyrimidine tract binding protein

“…Raver1 recruitment is essential for effective repressor function of PTB in certain pre-mRNAs, for example for the splicing repression of a-tropomyosin exon 3, while in other systems PTB alone is sufficient. Furthermore, tissue-specific paralogs of PTB exist, such as neuronally enriched PTB [9,40] (nPTB), the smooth muscle-specific variant (smPTB) [41] or ROD1 the variant found in rat hematopoietic cell [42], which appear to have different effects on splicing regulation [9,41]. The second most studied function of PTB is its role in internal ribosomal entry site (IRES)-mediated translation initiation of both cellular and viral mRNAs.…”

“…PTB is one of the most frequently encountered ITAFs and was found to be implicated in IRES-mediated translation of several Figure 1. Polypyrimidine tract binding protein (PTB) is a ubiquitous regulator of alternative splicing that influences different types of alternative-splicing events (a) PTB represses the N1 "cassette-exon" in the c-src pre-mRNA [7,9,10,103]. (b) PTB represses the "mutually exclusive" SM exon of the a-actinin mRNA [12,13].…”

“…and RRM4 are highly conserved across species, as well as in PTB paralogs [41,42] like nPTB [9,40] and hence it seems likely that this topology is functionally significant. The NMR structures of free RRM1 and RRM2 have been assessed in another study [82].…”

Control of alternative pre-mRNA splicing by Ca++ signals

“…The relative levels of positive and negative regulatory factors determine the generation of variant mRNA isoforms in specific cell types [73][74][75]. Brainenriched splicing factors include Nova-1 and -2 [62][63][64][65][66], FOX-1 and -2 [54-61], PTBP2 (nPTB) and neuronal Hu proteins [76][77][78][79][80][81][82][83].…”