Hostname: page-component-848d4c4894-mwx4w Total loading time: 0 Render date: 2024-06-14T10:37:08.523Z Has data issue: false hasContentIssue false

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

Published online by Cambridge University Press:  01 March 1999

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208, USA Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794-5222, USA
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208, USA
Get access


The rat β-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.

Research Article
1999 RNA Society

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)