Exonic splicing enhancers contribute to the use of both 3' and 5' splice site usage of rat beta-tropomyosin pre-mRNA

Selvakumar, M., Helfman, D. M. (March 1999) Exonic splicing enhancers contribute to the use of both 3' and 5' splice site usage of rat beta-tropomyosin pre-mRNA. Rna, 5 (3). pp. 378-94. ISSN 1355-8382 (Print)

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URL: http://www.ncbi.nlm.nih.gov/pubmed/10094307

Abstract

The rat beta-tropomyosin gene encodes two tissue-specific isoforms that contain the internal, mutually exclusive exons 6 (nonmuscle/smooth muscle) and 7 (skeletal muscle). We previously demonstrated that the 3' splice site of exon 6 can be activated by introducing a 9-nt polyuridine tract at its 3' splice site, or by strengthening the 5' splice site to a U1 consensus binding site, or by joining exon 6 to the downstream common exon 8. Examination of sequences within exons 6 and 8 revealed the presence of two purine-rich motifs in exon 6 and three purine-rich motifs in exon 8 that could potentially represent exonic splicing enhancers (ESEs). In this report we carried out substitution mutagenesis of these elements and show that some of them play a critical role in the splice site usage of exon 6 in vitro and in vivo. Using UV crosslinking, we have identified SF2/ASF as one of the cellular factors that binds to these motifs. Furthermore, we show that substrates that have mutated ESEs are blocked prior to A-complex formation, supporting a role for SF2/ASF binding to the ESEs during the commitment step in splicing. Using pre-mRNA substrates containing exons 5 through 8, we show that the ESEs within exon 6 also play a role in cooperation between the 3' and 5' splice sites flanking this exon. The splicing of exon 6 to 8 (i.e., 5' splice site usage of exon 6) was enhanced with pre-mRNAs containing either the polyuridine tract in the 3' splice site or consensus sequence in the 5' splice site around exon 6. We show that the ESEs in exon 6 are required for this effect. However, the ESEs are not required when both the polyuridine and consensus splice site sequences around exon 6 were present in the same pre-mRNA. These results support and extend the exon-definition hypothesis and demonstrate that sequences at the 3' splice site can facilitate use of a downstream 5' splice site. In addition, the data support the hypothesis that ESEs can compensate for weak splice sites, such as those found in alternatively spliced exons, thereby providing a target for regulation.

Item Type: Paper
Uncontrolled Keywords: Animals Binding Sites/genetics Cell Line Cross-Linking Reagents DNA-Binding Proteins/genetics Exons/ genetics Mutagenesis/genetics Nuclear Proteins/genetics RNA Precursors/ genetics RNA Splicing/ genetics Rats Research Support, U.S. Gov't, P.H.S. Spliceosomes/genetics Tropomyosin/ genetics Ultraviolet Rays
Subjects: organs, tissues, organelles, cell types and functions > cell types and functions > cell types > cell line
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > cell line
organs, tissues, organelles, cell types and functions > cell types and functions > cell types > cell line

bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > DNA binding protein
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > exons
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > mutations > mutagenesis
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > RNA binding protein
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 > spliceosome complex
CSHL Authors:
Communities: CSHL labs > Helfman lab
Depositing User: Kathleen Darby
Date: March 1999
Date Deposited: 30 Apr 2014 13:55
Last Modified: 05 May 2014 16:56
PMCID: PMC1369767
Related URLs:
URI: http://repository.cshl.edu/id/eprint/29803

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