scholarly journals 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

2006 ◽  
Vol 26 (23) ◽  
pp. 8755-8769 ◽  
Author(s):  
Jérôme Saulière ◽  
Alain Sureau ◽  
Alain Expert-Bezançon ◽  
Joëlle Marie
Keyword(s):  
Branch Point ◽  
Early Stage ◽  
Binding Protein ◽  
Polypyrimidine Tract ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein ◽  
Nuclear Extracts ◽  
Nonmuscle Cells ◽  
Labeled Rna

ABSTRACT Splicing of exon 6B from the β-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.

scholarly journals Polypyrimidine Tract Binding Protein Functions as a Repressor To Regulate Alternative Splicing of α-Actinin Mutally Exclusive Exons

1999 ◽  
Vol 19 (4) ◽  
pp. 2699-2711 ◽  
Author(s):  
Justine Southby ◽  
Clare Gooding ◽  
Christopher W. J. Smith
Keyword(s):  
Branch Point ◽  
Splice Site ◽  
Binding Protein ◽  
Regulatory Elements ◽  
Regulatory Sequences ◽  
Branch Points ◽  
Polypyrimidine Tract ◽  
Polypyrimidine Tract Binding ◽  
Polypyrimidine Tract Binding Protein

ABSTRACT The smooth muscle (SM) and nonmuscle (NM) isoforms of α-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat α-actinin genomic clone encompassing the mutually exclusive exons was isolated and sequenced. The SM exon was found to utilize two branch points located 382 and 386 nucleotides (nt) upstream of the 3′ splice site, while the NM exon used a single branch point 191 nt upstream. Mutually exclusive splicing arises from the proximity of the SM branch points to the NM 5′ splice site, and this steric repression could be relieved in part by the insertion of spacer elements. In addition, the SM exon is repressed in non-SM cells and extracts. In vitro splicing of spacer-containing transcripts could be activated by (i) truncation of the transcript between the SM polypyrimidine tract and exon, (ii) addition of competitor RNAs containing the 3′ end of the actinin intron or regulatory sequences from α-tropomyosin (TM), and (iii) depletion of the splicing extract by using biotinylated α-TM RNAs. A number of lines of evidence point to polypyrimidine tract binding protein (PTB) as the trans-acting factor responsible for repression. PTB was the only nuclear protein observed to cross-link to the actinin RNA, and the ability of various competitor RNAs to activate splicing correlated with their ability to bind PTB. Furthermore, repression of α-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA could be specifically restored by the addition of recombinant PTB. Thus, α-actinin mutually exclusive splicing is enforced by the unusual location of the SM branch point, while constitutive repression of the SM exon is conferred by regulatory elements between the branch point and 3′ splice site and by PTB.

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

2000 ◽  
Vol 20 (20) ◽  
pp. 7463-7479 ◽  
Author(s):  
Vadim Markovtsov ◽  
Julia M. Nikolic ◽  
Joseph A. Goldman ◽  
Christoph W. Turck ◽  
Min-Yuan Chou ◽  
...  
Keyword(s):  
Binding Protein ◽  
Regulatory Protein ◽  
Regulatory Elements ◽  
Specific Protein ◽  
Regulatory Sequence ◽  
Polypyrimidine Tract ◽  
Tissue Specific ◽  
Polypyrimidine Tract Binding ◽  
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.

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