MECHANISMS OF PRE-MRNA SPLICING IN HIGHER EUKARYOTES

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The long term objective of our research is to understand the mechanisms of pre-mRNA splicing in higher eukaryotes. Most pre-mRNAs in metazoa are highly complex, containing multiple introns that must be precisely excised in order to generate functional mRNA. Splicing defects are responsible for many genetic diseases, and splicing is a critical step in the life cycle of virtually all viruses. Thus, elucidating the mechanisms of splicing is a problem of fundamental importance. This research proposal is focused on achieving a detailed understanding of the early steps in mammalian spliceosome assembly. These steps are especially important because the mechanisms responsible for identifying each of the splice sites and for bringing together the appropriate pairs of 5' and 3' splice sites in both constitutively and alternatively spliced pre-mRNAs operate at this time. Spliceosome assembly is initiated by the ATP-independent formation of the E complex followed by formation of the ATP-dependent A complex. In previous work these complexes were purified and found to contain several key components of the splicing machinery, including U1 and U2 snRNPs, the non-snRNP splicing factor U2AF, and the SR family of splicing proteins. The general goal of the first aim of this proposal is to understand how each these components interact with one another and with specific sequence elements in the pre-mRNA at distinct times during assembly of the E and A complexes. The second specific aim is to reconstitute the U2 snRNP particle using wild-type and mutant derivatives of recombinant U2 snRNP- associated proteins. The reconstituted snRNP will then be added back to splicing extracts depleted of U2 snRNP in order to determine the specific functions of each of the U2 snRNP components in both spliceosome assembly and the catalytic steps of the splicing reaction. In the third specific aim, a working model has been formulated to explain the molecular basis for splice-site selection in constitutively spliced pre-mRNAs. One of the key features of the model is that exons contain one or more specific sequence elements, designated "exonic selectors", that promote the use of the adjacent 5' or 3' splice site. The exonic selectors are proposed to function via differential interactions with members of the SR family of splicing proteins. Another central feature of the model is that factors bound to three distinct elements in the pre-mRNA, the exonic selector, and the 5' and 3' splice sites flanking an exon, reciprocally promote each other binding to pre- mRNA. The central aspects of the model will be tested by a combination of identifying the binding sites of SR proteins within the exons in functional E complex and determining whether these binding sites affect splice-site selection. In addition, studies used purified factors will be carried out to determine whether factors that bind to exonic selectors and the flanking 5' and 3' splice sites influence one anothers binding.