Polypyrimidine Tract Binding Protein Prevents Activity of an Intronic Regulatory Element That Promotes Usage of a Composite 3′-Terminal Exon

Vincent Anquetil,Caroline Le Sommer,Agnès Méreau,Sandra Hamon,Hubert Lerivray,Serge Hardy

doi:10.1074/jbc.m109.029314

Abstract

Alternative splicing of 3'-terminal exons plays a critical role in gene expression by producing mRNA with distinct 3'-untranslated regions that regulate their fate and their expression. The Xenopus alpha-tropomyosin pre-mRNA possesses a composite internal/3'-terminal exon (exon 9A9') that is differentially processed depending on the embryonic tissue. Exon 9A9' is repressed in non-muscle tissue by the polypyrimidine tract binding protein, whereas it is selected as a 3'-terminal or internal exon in myotomal cells and adult striated muscles, respectively. We report here the identification of an intronic regulatory element, designated the upstream terminal exon enhancer (UTE), that is required for the specific usage of exon 9A9' as a 3'-terminal exon in the myotome. We demonstrate that polypyrimidine tract binding protein prevents the activity of UTE in non-muscle cells, whereas a subclass of serine/arginine rich (SR) proteins promotes the selection of exon 9A9' in a UTE-dependent way. Morpholino-targeted blocking of UTE in the embryo strongly reduced the inclusion of exon 9A9' as a 3'-terminal exon in the endogenous mRNA, demonstrating the function of UTE under physiological circumstances. This strategy allowed us to reveal a splicing pathway that generates a mRNA with no in frame stop codon and whose steady-state level is translation-dependent. This result suggests that a non-stop decay mechanism participates in the strict control of the 3'-end processing of the alpha-tropomyosin pre-mRNA.

Highlights

Association pour la Recherche contre le Cancer. 2 Present address: Dept. of Molecular Genetics and Microbiology and Center for RNA Biology, Duke University Medical Center, Durham, NC, 27710. 3 To whom correspondence should be addressed
upstream terminal exon enhancer (UTE), an Intronic Regulatory Element Required for the Splicing of Exon 9A9Ј as a 3Ј-Terminal Exon—We previously demonstrated that the major branch point used in the myotome to splice exon 9A9Ј as a 3Ј-terminal exon is localized 274 nucleotides upstream of the AG dinucleotide
Mutation of this branch site or of the associated 21-nucleotide-long pyrimidine tract resulted in a strong skipping of exon 9A9Ј in a minigene-derived transcript expressed in the myotome [17]

Summary

Introduction

Association pour la Recherche contre le Cancer. 2 Present address: Dept. of Molecular Genetics and Microbiology and Center for RNA Biology, Duke University Medical Center, Durham, NC, 27710. 3 To whom correspondence should be addressed. Specific splicing of exon 9A9Ј as a 3Ј-terminal exon by the selection of the ␣e poly(A) site present within this exon produces the ␣7 mRNA that encodes muscle ␣-TM [15] (see Fig. 1).

Results

Conclusion