Alternative splicing of beta-tropomyosin pre-mRNA: cis-acting elements and cellular factors that block the use of a skeletal muscle exon in nonmuscle cells

Guo, W., Mulligan, G. J., Wormsley, S., Helfman, D. M. (November 1991) Alternative splicing of beta-tropomyosin pre-mRNA: cis-acting elements and cellular factors that block the use of a skeletal muscle exon in nonmuscle cells. Genes Dev, 5 (11). pp. 2096-107. ISSN 0890-9369 (Print)0890-9369 (Linking)

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Abstract

The rat beta-tropomyosin (beta-TM) gene encodes both skeletal muscle beta-TM and fibroblast TM-1 by an alternative RNA-splicing mechanism. This gene contains 11 exons. Exons 1-5, 8, and 9 are common to all mRNAs expressed from the gene. Exons 6 and 11 are used in fibroblasts as well as smooth muscle cells, whereas exons 7 and 10 are used in skeletal muscle cells. In this study we have carried out an extensive mutational analysis to identify cis-acting elements that block the use of the skeletal muscle-specific exon 7 in nonmuscle cells. These studies localize the critical elements for regulated alternative splicing to sequences within exon 7 and the adjacent upstream intron. In addition, mutations that inactivate the 5'- or 3'-splice sites of exon 6 do not result in the use of the skeletal muscle-specific exon 7 in nonmuscle cells, suggesting that splice-site selection in vivo is not regulated by a simple cis-acting competition mechanism but, rather, by a mechanism that inhibits the use of exon 7 in certain cellular environments. In support of this hypothesis we have identified sequence-specific RNA-binding proteins in HeLa cell nuclear extracts using native gel electrophoresis and binding competition assays. Mutations in the pre-mRNA that result in the use of the skeletal muscle exon in vivo also disrupt the binding of these proteins to the RNA in vitro. We propose that the binding of these proteins to the pre-mRNA is involved in regulated alternative splicing and that this interaction is required for blocking the use of the skeletal muscle exon in nonmuscle cells.

Item Type:	Paper
Uncontrolled Keywords:	Base Sequence Exons/*genetics Gene Expression Regulation/*genetics HeLa Cells Humans Introns/genetics Molecular Sequence Data Muscles/metabolism Mutagenesis, Site-Directed Plasmids/genetics RNA Precursors/*metabolism RNA Processing, Post-Transcriptional/genetics RNA Splicing/*genetics Transfection/genetics Tropomyosin/*genetics
Subjects:	bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > Alternative Splicing bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > pre-mRNA bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNA splicing bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > tropomyosin
CSHL Authors:	Guo, Wei Helfman, David M. Mulligan, George Wormsley, Steven
Communities:	CSHL labs > Helfman lab
Depositing User:	Matt Covey
Date:	November 1991
Date Deposited:	14 Jan 2016 16:44
Last Modified:	03 Nov 2017 20:51
Related URLs:	Publisher
URI:	https://repository.cshl.edu/id/eprint/32038

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