Mutations in RRM4 uncouple the splicing repression and RNA-binding activities of polypyrimidine tract binding protein

Abstract

The polypyrimidine tract binding protein (PTB, or hnRNP I) contains four RNA-binding domains of the ribonucleoprotein fold type (RRMs 1, 2, 3, and 4), and mediates the negative regulation of alternative splicing through sequencespecific binding to intronic splicing repressor elements. To assess the roles of individual RRM domains in splicing repression, a neural-specific splicing extract was used to screen for loss-of-function mutations that fail to switch splicing from the neural to nonneural pathway. These results show that three RRMs are sufficient for wild-type RNA binding and splicing repression activity, provided that RRM4 is intact. Surprisingly, the deletion of RRM4, or as few as 12 RRM4 residues, effectively uncouples these functions. Such an uncoupling phenotype is unique to RRM4, and suggests a possible regulatory role for this domain either in mediating specific RNA contacts, and/or contacts with putative splicing corepressors. Evidence of a role for RRM4 in anchoring PTB binding adjacent to the branch site is shown by mobility shift and RNA footprinting assays.

Alternative Splicing of the Multidrug Resistance Protein 1/ATP Binding Cassette Transporter Subfamily Gene in Ovarian Cancer Creates Functional Splice Variants and Is Associated with Increased Expression of the Splicing Factors PTB and SRp20

“…As a splicing factor, PTB has been shown to repress exon inclusion in a number of genes and, thus, induce exon skipping. Genes regulated negatively by PTB in pre-mRNA splicing include ␣-and ␤-tropomyosin (33-35), FGF-R1 (36), FGF-R2 (37,38), c-src (39), GABA␥2 (40), fibronectin (41), caspase-2 (42), and ␣-actinin (43). Our results suggest that the MRP1 gene could be another target regulated by PTB.…”

Structure-function relationships of the polypyrimidine tract binding protein

“…The distribution of PTB binding sites within introns has led to several proposed models of PTB action. For example, PTB could interfere with spliceosome assembly by binding to the branch point pyrimidine tract and therefore directly sequestering the branch-point or competing with U2AF, an essential component of the constitutive splicing machinery [2,15,22,32]. In most cases, however, the exons silenced by PTB are flanked by PTB binding sites on both adjacent introns, and mutations of upstream PTB binding sequences have been shown to reduce binding of PTB to the downstream sites [7].…”

“…65,2008 Review Article 521 to function through an interaction with PTB, and might facilitate the identification of novel PTB targets. For example, many studies identified PTB binding sites by boundary analyses, gel shift experiments and cross-linking [10,15,22,32]. In light of the complex structures [85], these findings can be more easily interpreted.…”

Next-generation SELEX identifies sequence and structural determinants of splicing factor binding in human pre-mRNA sequence

“…One such repressor is the abundant polypyrimidine tract binding protein (PTB), a well-studied member of the hnRNP family (Perez et al 1997a;Liu et al 2002;Shen et al 2004;Wollerton et al 2004;Amir-Ahmady et al 2005;Sharma et al 2005;Spellman and Smith 2006;Boutz et al 2007;Matlin et al 2007;Sawicka et al 2008). The simplest model of PTB-mediated silencing is through competition with U2AF65 for the binding of the polypyrimidine tract (Singh et al 1995;Liu et al 2002;Sauliere et al 2006;Matlin et al 2007).…”