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 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 (la, 9, 13, 34) or the secondary structure of the region around splice sites (10,11,25,38,39,46) affects splice site selection. In addition, there are numerous reports that suggest a role for specific exon sequences in splicing (4,6,12,15,16,18,20,24,26,27,32,33,40,41,45,48,49).We have previously shown that a purine-rich sequence located within the last exon, M2, of the mouse immunoglobulin p, (IgM) gene plays an essential role in the splicing of this gene (47,48). We found that this sequence functions as a splicing enhancer: it stimulated splicing of a distant intron that is present in the upstream region. This stimulatory effect was observed even with introns derived from different genes (48). We also found that this sequence promotes the formation of the early splicing complex (48). These results show that the sequence that we identified within IgM exon M2 represents a novel element involved in splice site selection. We designated this sequence element ERS (exon recognition sequence).Moreover, we have found that several exon sequences whose mutation or deletion affects splice site selection contain purine-rich sequences similar to the ERS of IgM exon M2 (IgM-ERS) (see Table 1 in reference 48). The examples include the avian sarcoma-leukosis virus (ASLV) gene (12,21), the chicken cardiac troponin T (cTNT) gene (4,5,49), the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene (41), the bovine growth hormone (BGH) gene (15), the rat beta-tropomyosin gene (16), the fibronectin gene (27), and the neural cell adhesion molecule (NCAM) gene (42). This observation raised the possibility that the purinerich sequences of these genes are other examples of ERS and that the effects of exon mutations are ascribed to the loss of the enhancer activity of ERS. In this regard, Xu et al. recently reported that both the purine-rich sequence of the cTNT exon and the synthetic GAAGAGGAGG repeat sequence facilitate splicing of a heterologous intron in in vivo experiments (49). This finding is consistent with our notion

Exonic splicing enhancers in fission yeast: functional conservation demonstrates an early evolutionary origin

“…In vitro splicing assays have shown that deletions or substitutions of the (GAR) n repeats found in ASLV, IgM, and related ESEs diminish or abolish their ability to promote splicing of an upstream intron (Katz and Skalka 1990;Fu et al 1991;Watakabe et al 1993;Xu et al 1993;Dirksen et al 1994;Tanaka et al 1994). To assess the dependence of ESE activity in S. pombe upon the purine-rich repeats, we constructed two mutant versions of the ASLV ESE located downstream from the 3Ј-splice site in the cdc2-I2 9R minigene (Fig.…”

Modulation in vitro of H-ras oncogene expression by trans-splicing

“…The (ras)IDX contains no (GAR) n repeats in its sequence. Therefore, the ESE sequence of the ASLV (Tanaka et al, 1994) was added to the (ras)IDX, 50 nt away from its 3'SS, in order to determine whether this sequence could activate trans-splicing of the (ras)IDX. This molecule was named N pre-mRNA.…”

“…For (M) plasmid L was linearized with PstI and transcribed with T7 RNA polymerase. For (N) the avian sarcoma-leukosis virus (ASLV) enhancer was obtained from pSP72-ASLV (env 3' exon) (Tanaka et al, 1994) that had been cut with XbaI and HindIII, and the fragment was subcloned in Bluescript SK (7) plasmid that had itself previously been cut with XbaI and HindIII. This plasmid, named pre-ASLV, was ®nally cut with XbaI.…”

A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer.

“…Exonic splicing enhancers are sequences, found in certain exons, that can stimulate spliceosome assembly at the upstream 3' splice site of the exon (Fu et al 1991;Lavigueur et al 1993;Sun et al 1993;Watakabe et al 1993;Caputi et al 1994;Dirksen et al 1994;Staknis and Reed 1994;Tanaka et al 1994;Tian and Maniatis 1994;Ramchatesingh et al 1995;Wang et al 1995). Exonic enhancers are usually purine-rich sequences whose ac-…”