Activation of α-Tropomyosin Exon 2 Is Regulated by the SR Protein 9G8 and Heterogeneous Nuclear Ribonucleoproteins H and F

Abstract

The inclusion of exons 2 and 3 of α-tropomyosin is governed through tissue-specific alternative splicing. These exons are mutually exclusive, with exon 2 included in smooth muscle cells and exon 3 included in nearly all other cell types. Several cis-acting sequences contribute to this splicing decision: the branchpoints and pyrimidine tracts upstream of both exons, UGC-repeat elements flanking exon 3, and a series of purine-rich enhancers in exon 2. Previous work showed that proteins rich in serine-arginine (SR) dipeptides act through the exon 2 enhancers, but the specific proteins responsible for such activation remained unknown. Here we show that a 35-kDa member of the SR protein family, 9G8, can activate the splicing of α-tropomyosin exon 2. Using RNA affinity chromatography and cross-linking competition assays, we also demonstrate that the heterogeneous nuclear ribonucleoproteins (hnRNPs) H and F bind to and compete for the same elements. Overexpression of hnRNPs H and F blocked 9G8-mediated splicing both in vivo and in vitro, and small interfering RNA-directed depletion of H and F led to an increase in exon 2 splicing. These data suggest that the activation of exon 2 is dependent on the antagonistic activities of 9G8 and hnRNPs H and F.

Supplemental material for this article may be found at http://mcb.asm.org/.

We thank Doug Black, Brenton Graveley, James Stévenin, Adrian Krainer, Robert Lafyatis, and Tracey Rouault for kindly providing expression constructs, antibodies, cDNA clones, and protocols. We also thank David Friedman and the Vanderbilt Proteomics Laboratory.

This study was supported by an NIH grant to J.G.P. (GM 62487) and the Vanderbilt Mechanisms of Vascular Disease Training Grant (NIH 5 T32 HL07751).