Activation of alpha-tropomyosin exon 2 is regulated by the SR protein 9G8 and heterogeneous nuclear ribonucleoproteins H and F.
The inclusion of exons 2 and 3 of alpha-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 alpha-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.
Crawford
J Barrett
JB
Department of Biological Sciences, Box 1820 Station B, Vanderbilt University, Nashville, TN 37235, USA.
Patton
James G
JG
D017398:Alternative Splicing; D002460:Cell Line; D005091:Exons; D006367:HeLa Cells; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H / Q000502:physiology*; D006801:Humans; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D014335:Tropomyosin / Q000235:genetics* / Q000378:metabolism*
D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D016601:RNA-Binding Proteins; D014335:Tropomyosin; D000068103:Serine-Arginine Splicing Factors