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PMID: 10094307 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

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

RNA (New York, N.Y.) ·Vol. 5 ·No. 3 ·1999-03-00 ·Pages 378-94

Selvakumar M, Helfman DM

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.

MeSH Terms
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 RNA-Binding Proteins Rats Serine-Arginine Splicing Factors Spliceosomes/genetics Tropomyosin/genetics Ultraviolet Rays
Chemicals
Cross-Linking Reagents DNA-Binding Proteins Nuclear Proteins RNA Precursors RNA-Binding Proteins Tropomyosin Serine-Arginine Splicing Factors
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Selvakumar M
Cold Spring Harbor Laboratory, New York 11724-2208, USA.
Helfman D M
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1355-8382
DOI
10.1017/s1355838299981050
Published
1999-03-00
Pages
378-94
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC1369767
Subset
IM
Grants
NIGMS NIH HHS · GM43049 · United States
External Links
PubMed source DOI: 10.1017/s1355838299981050 PMC Full Text View journal details
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