Identification of Proteins That Interact with Exon Sequences, Splice Sites, and the Branchpoint Sequence during Each Stage of Spliceosome Assembly

Abstract

We have carried out a systematic analysis of the proteins that interact with specific intron and exon sequences during each stage of mammalian spliceosome assembly. This was achieved by site-specifically labeling individual nucleotides within the 5 and 3 splice sites, the branchpoint sequence (BPS), or the exons with 32 P and identifying UV-cross-linked proteins in the E, A, B, or C spliceosomal complex. Significantly, two members of the SR family of splicing factors, which are known to promote E-complex assembly, cross-link within exon sequences to a region ϳ25 nucleotides upstream from the 5 splice site. At the 5 splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5 200, was detected in both the B and C complexes. As observed in yeast cells, U5200 also cross-links to intron/exon sequences at the 3 splice site in the C complex and may play a role in aligning the 5 and 3 exons for ligation. With label at the branch site, we detected three distinct proteins, designated BPS 72, BPS 70 , and BPS 56 , which replace one another in the E, A, and C complexes. Another dynamic exchange was detected with pre-mRNA labeled at the AG dinucleotide of the 3 splice site. In this case, a protein, AG 100 , cross-links in the A complex and is replaced by another protein, AG 75 , in the C complex. The observation that these proteins are specifically associated with critical pre-mRNA sequence elements in functional complexes at different stages of spliceosome assembly implicates roles for these factors in key recognition events during the splicing pathway.During pre-mRNA splicing, a series of highly dynamic spliceosomal complexes, consisting of multiple protein and small nuclear RNA (snRNA) components, assemble on pre-mRNA in the order E 3 A 3 B 3 C (for reviews, see references 5, 12, 20, 22, 27, and 31). The sequence elements required for assembly of these complexes are located at the 5Ј and 3Ј splice sites and at the branch site, and exon sequences affect the recognition of both splice sites. In metazoans, all of the elements involved in splicing are weakly conserved. Additional specificity is derived from strict constraints on the locations of these elements relative to one another, which presumably allows for complex networks of RNA-protein, protein-protein, and RNA-RNA interactions between factors bound to each element. Studies of the sequences required for splicing indicate that all of the critical elements are recognized multiple times during the splicing pathway. This proofreading, combined with the vast number of specific interactions established, is most likely the key to achieving high fidelity in the splicing reaction. A detailed understanding of the splicing mechanism requires identifying all of the factors that recognize each of the elements during the different stages of spliceosome assembly and understanding how these factors interact with both the pre-mRNA and one another.A great deal of progress in identifying key recognition factors has come from a combination of genetic and...

5′ exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly

“…Identification of the p25 cross-link as SRp20 is consistent with a previous report from the Reed laboratory (Chiara et al 1996) that SRp20 (and SRp30) could be cross-linked in the E/CC complex at positions −31 and −26 relative to the 5Ј splice site of AdML pre-mRNA. We similarly reported that SRp20 cross-links to position −2 of PIP pre-mRNA independent of splicing (Le Hir et al 2000a).…”

“…The p220 that cross-linked to benzophenone at position −7 is most likely hPrp8p, as we and others have previously demonstrated that hPrp8p cross-links strongly near the end of the 5Ј exon and at the 5Ј splice site (Chiara et al 1996 and references therein; Reyes et al 1999;Le Hir et al 2000a). Likewise, the p170 that cross-linked only after exon ligation may well be SRm160-using a different antiserum from that used here (see Materials and Methods), we have previously shown that SRm160 can become cross-linked to benzophenone incorporated adjacent to the exon-exon junction of spliced mRNA (Le Hir et al 2000a).…”

Distinct domains of splicing factor Prp8 mediate different aspects of spliceosome activation

“…Prp8 is a U5 RNA-associated protein that contacts both the 5Ј and 3Ј splice sites of the intron (12)(13)(14)(15) as well as U6 RNA (16). Prp8 has been implicated in regulation of the second transesterification reaction (17)(18)(19)(20); our results suggest that Prp8 also functions in the first catalytic step of splicing.…”

Regulation of Alternative Splicing of CD45 by Antagonistic Effects of SR Protein Splicing Factors

“…Several independent studies identified a SRp20-binding sequence with enhancer activity, all sharing the degenerate sequence CUC(U/ G)UC(C/T) (51)(52)(53)(54). In addition, application of the so-called SELEX strategy yielded SRp20-specific sequences with the consensus CA/UA/UC (55).…”