Polypurine Sequences within a Downstream Exon Function as a Splicing Enhancer

Abstract

We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin mu gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin mu gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection.

Functional crosstalk between exon enhancers, polypyrimidine tracts and branchpoint sequences

“…Even in Saccharomyces cerevisiae , where there appears to be little or no alternative splicing and pyrimidine tracts are often unrecognizable, increasing the polypyrimidine content can increase the use of adjacent 3′ splice sites (Parker and Patterson, 1987; Patterson and Guthrie, 1991). Exon sequences have also been shown to act in combination with other splicing signals to increase the efficiency of splicing and enable greater regulation (Hoshijima et al ., 1991; Lavigueur et al ., 1993; Sun et al ., 1993; Watakabe et al ., 1993; Xu et al ., 1993; Tanaka et al ., 1994; Tian and Maniatis, 1994; Humphrey et al ., 1995). Generally, these so‐called splicing enhancer elements are purine rich, but non‐purine‐rich sequences have also been shown to act as splicing enhancers (Staknis and Reed, 1994; Tian and Kole, 1995; Coulter et al ., 1997).…”

A Splicing Variant of the RON Transcript Induces Constitutive Tyrosine Kinase Activity and an Invasive Phenotype

“…In general, it has been shown that the selection of a splice site depends not only on its own sequence but also on the context around that sequence (28). Regarding this point, exons that are intrinsically poor splicing substrates, because of small size or weak flanking splice sites, have been proposed to contain positive-acting splicing elements, referred to as exonsplicing enhancers (17,42,48). Canonical exon-splicing enhancer sequences were, however, not easily identifiable in the 147-bp cassette-exon region.…”

Splicing fidelity, enhancers, and disease

“…Similarly, if purine content is the major factor, replacement of one purine-rich enhancer sequence of a certain length with another equally purine-rich sequence of the same length should result in similar splicing outcomes. But again, this is not the case (47). These examples highlight the importance of specific sequences, in addition to purinecontent, in defining enhancer elements.…”

“…Exonic enhancers direct U2AF and U2 binding to the 3′ splice site and U1 binding to the 5′ splice site, respectively. For bona fide enhancers it appears that the specific sequence is more important than the overall purine content (47). If the purine content was a more important variable than the specific sequence, then changes to the sequence that do not affect purine content would be expected to have no consequence.…”