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 ³²P 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 ~25 nucleotides upstream from the 5′ splice site. At the 5′ splice site, cross-linking of the U5 small nuclear ribonucleoprotein particle protein, U5²⁰⁰, 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 BPS⁷², BPS⁷⁰, and BPS⁵⁶, 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, AG¹⁰⁰, cross-links in the A complex and is replaced by another protein, AG⁷⁵, 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.