Regulation of U1 snRNP/5' Splice Site Interactions During pre-mRNA Splicing

Ohio State University Research Foundation -Do Not Use, Columbus OH

Investigators

Abstract

Abstract Many eukaryotic genes consist of coding sequences (exons) separated by intervening sequences (introns) that are removed from precursor RNAs (Pre-RNAs) by RNA splicing. Pre-messenger RNA (Pre-mRNA) splicing takes place in the spliceosome, a macromolecular complex composed of a large number of proteins and five small nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6. The spliceosome assembles and rearranges in a highly ordered and stepwise manner, which, at the molecular level, corresponds to a series of sequential changes of RNA-RNA interactions. Strikingly, many of these interactions are mutually exclusive, i.e. the formation of one requires the disruption of another. For example, the 5' splice site (5'SS) is initially recognized by U1 snRNA via base-pairing interaction, which is subsequently switched to a mutually exclusive base-pairing interaction with U6 snRNA. "RNA unwindases" (or RNA helicases) are thought to govern these RNA rearrangements. This project continues to investigate the function of Prp28p, an RNA unwindase, in nuclear pre-mRNA splicing in the budding yeast Saccharomyces cerevisiae. Recently, it was discovered that mutations in the U1-C protein, which is important to the stable binding of U1 snRNP to 5'SS, can bypass the otherwise essential Prp28p in pre-mRNA splicing and that, in such a system, a loss-of-function mutation in PRP8 can re-impose the demand of Prp28p. Considering the potential role of Prp28p in RNA unwinding, it was therefore proposed that Prp28p and Prp8p are functionally antagonistic to U1-C and that they are likely to work in concert to dissociate U1 snRNP from 5'SS. Remarkably, these conclusions are in good agreement with two recent reports regarding the roles of Prp28p and Prp8p in controlling the switch of U1 for U6 at 5'SS. This research thus aims to expand and sharpen our current understanding of this control mechanism by exploiting the unique bypass suppressor system both genetically and biochemically. Specific aims are: (1) To continue to identify and characterize components crucial to the U1 snRNP/5'SS interaction and to map regions in Prp8p involved in this interaction; (2) To develop a temperature-dependent in vitro splicing system for testing the predictions stemming from the previous genetic work and for investigating the functions of Prp28p and Prp8p. Many eukaryotic genes consist of coding sequences (exons) separated by intervening sequences (introns) that are removed from precursor RNAs (Pre-RNAs) by RNA splicing. The splicing step is often used to produce tissue-specific or developmentally regulated protein isoforms. Further, defective RNA splicing has been implicated in several genetic diseases and in the life cycle of a wide variety of viruses. Studies on the mechanism of RNA splicing are therefore of fundamental importance to our understanding of eukaryotic gene structure and gene expression. This research focuses on characterizing a critical regulatory circuit in pre-mRNA splicing, in which several specific proteins (termed Prp28p, Prp8p, U1-C), and possibly others, jointly regulate critical interactions within the splicing apparatus.