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

Abstract

The smooth muscle (SM) and nonmuscle (NM) isoforms of ␣-actinin are produced by mutually exclusive splicing of an upstream NM exon and a downstream SM-specific exon. A rat ␣-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 spacercontaining 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 ␣-tropomyosin (TM), and (iii) depletion of the splicing extract by using biotinylated ␣-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 ␣-actinin splicing in the nuclear extracts depleted of PTB by using biotinylated RNA could be specifically restored by the addition of recombinant PTB. Thus, ␣-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.Many eukaryotic genes employ alternative splicing as a means of generating protein diversity. This differential incorporation of exons into the mature RNA is often under developmental and/or tissue-specific control and enables the cell to tailor the protein to suit its own particular requirements (61, 67). The basic splicing mechanism involves a two-step process which takes place in a ribonucleoprotein complex called a spliceosome and results in adjacent exons being joined together with the intron between released in the form of a lariat (reviewed in references 1 and 57). There is a further level of complexity in alternative splicing in that different combinations of 5Ј and 3Ј splice sites are ligated. The mechanisms that determine which splice sites are utilized and how this is regulated in different cell types or developmental stages have still not been precisely defined. Much progress has been made in identifying the cis-acting elements involved in alternative splicing, and the roles of some general factors have been demonstrated (1, 67). cis-Acting determinants that influence competing splicing pathways include the relative strengths of the competing 5Ј splice sites (e.g., 9, 78), branch point sequences (e.g., 53, 79), and polypyrimidine tracts...

A brain-enriched polypyrimidine tract-binding protein antagonizes the ability of Nova to regulate neuron-specific alternative splicing

“…This action of PTB is believed to be mediated by PTB binding to polypyrimidine-rich elements and blocking the assembly of a splicing competent complex in a manner analogous to the action of Sxl to block U2AF65 polypyrimidine tract binding (40). For example, PTB acts to repress smooth muscle-specific inclusion of alternatively spliced exons in the ␣-tropomyosin and ␣-actinin pre-mRNAs, most likely by binding to regulatory elements upstream of the 3Ј splice site (33,34,41). In neuronal cells, PTB inhibits inclusion of the n-src exon, in part by an action on 3Ј splice-site selection (20).…”

Polypyrimidine Tract-binding Protein Is Involved in Vivo in Repression of a Composite Internal/3′ -Terminal Exon of the Xenopus α-Tropomyosin Pre-mRNA

“…PTB has been implicated in the repression of alternative exons of many genes including ␣-fast tropomyosin, ␣-actinin, ␤-tropomyosin, and c-src (9,14,15,17). In most cases, PTB seems to reinforce the default splicing pattern through the repression of the tissue-specific exon inclusion.…”

“…For example, hnRNP A1 can antagonize the promotion of alternative exon usage that occurs through the binding of SR proteins to purine-rich exonic enhancer (7,8). The ubiquitously expressed polypyrimidine tract-binding protein (PTB designated also hnRNP I) has been implicated in the repression of several alternative exons including exon 7 of ␤-tropomyosin (9), exon N of GABA␥2 (10), exon 5 of N-methyl-D-aspartate receptor NR1 (11), exons IIIb and IIIc of FGF-R2 (12,13), exon N1 of c-src (14), exon SM of ␣-actinin (15), exon EIIIb of fibronectin (16), exon 3 of ␣-tropomyosin (17), and exon 9 of caspase-2 (18). PTB binds especially to pyrimidine-rich intronic splicing silencer elements present upstream or downstream of the alternative exon.…”

Structure-function relationships of the polypyrimidine tract binding protein

“…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]. (c) PTB regulates the choice of the 3'-terminal exon of the calcitonin/CGRP mRNA [104].…”