Using mouse immunoglobulin It (IgM) pre-mRNA as the model substrate for in vitro splicing, we have explored the role of exon sequences in splicing. We have found that deletion of the 5' portion of exon M2 of the IgM gene abolishes the splicing of its immediately upstream intron. Splicing was restored when a pudne-rich sequence found within the deleted region was reinserted into the deletion construct. This M2 exon sequence was able to stimulate the splicing of a heterologous intron of the Drosophila doublesex pre-mRNA that contains a suboptimal 3' splice site sequence. These results show that the IgM M2 exon sequence functions as a splicing enhancer. We found that the assembly of the early splicing complex is stimulated by the M2 exon sequence. In vitro competition experiments show that this stimulatory effect is mediated by the interaction of some trans-acting factors. Our results suggest that the U1 snRNP is one such factor. We propose that recognition of an enhancer exon sequence by the components of splicing machinery plays a vital role in the selection of splice sites, not only for the IgM pre-mRNA but for other pre-mRNAs. We designate such a sequence as exon recognition sequence {ERS).[Key Words: Splice site selection; splicing; exon recognition sequence; spliceosome assembly; U1 snRNP] Received September 28, 1992; revised version accepted December 28, 1992.Splicing of eukaryotic pre-mRNAs involves the accurate selection of the correct 5' and 3' splice sites. Previous studies have shown that conserved sequences around the 5' and 3' splice sites, including the site of lariat formation (branchpoint), serve as the major signal sequences in splice site determination (for review, see Krainer and Maniatis 1988;Green 1991). These sequence elements are recognized by splicing factors, which in turn trigger the formation of a multicomponent complex called the spliceosome (Brody and Abelson 1985;Frendewey and Keller 1985;Grabowski et al. 1985). Small nuclear ribonucleoprotein particles (snRNPs) U1, U2, and U4-U6, constitute the framework of the spliceosome. They bind to pre-mRNA in a stepwise manner: U1 and U2 snRNPs bind to the 5' splice site and the branchpoint sequence, respectively, to form an ATP-dependent complex (complex A or pre-spliceosome}. Subsequently, U4/U5/U6 snRNPs enter this complex and complete spliceosome (or complex B) formation. Determination of splice sites occurs early during spliceosome formation {Michaud and Reed 1991} and is followed by intron removal and exon ligation.The consensus for the 5' and 3' splice site sequences in higher eukaryotes has been determined by comparison of known intron sequences (Shapiro and Senapathy 1987}.The 5' consensus sequence is AG/GU(A/GIAGU, whereas the 3' consensus sequence contains a polypyrimidine stretch followed by CAG/G at the 3' splice site (YnNC~Cotresponding author.AG/G). The branchpoint sequence is also regarded as a part of the 3' consensus, although it is highly degenerate {Krainer and Maniatis 1988; Green 1991}. With the exception of the GU and AG ...

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

The expression of the IgM (immunoglobulin mu) heavy chain gene is known to be regulated at the post-transcriptional level. The two isoforms, the membrane-bound and secreted forms, are generated from the same gene by alternative processing at the 3' end of the primary transcript. The processing reactions involved are polyadenylation at the upstream poly(A) site (for the secreted form) and polyadenylation at the downstream poly(A) site coupled with splicing between exon C4 and exon M1 (for the membrane-bound form). The regulatory mechanism underlying these differential processing reactions is still not well understood. We investigated the splicing reaction between exon C4 and exon M1 in a HeLa nuclear extract using model transcripts containing the 5' and 3' splice sites of the C4-M1 intron. We found that the 3' splice site of the C4-M1 intron is sequestered in a stem-loop structure, which inhibits the splicing reaction in vitro. The inhibition by the stem-loop structure was also observed with a mouse lymphoma extract.

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.