We study the molecular mechanism of the pre-mRNA splicing reaction using the budding yeast Saccharomyces cerevisiae as a model system. RNA splicing is a complex process. The reaction occurs on the spliceosome, which is assembled via ordered interaction of five snRNAs and numerous protein factors to the pre-mRNA. The snRNAs play roles in the recognition and alignment of splice sites through base-paired interactions with the intron sequence and with each other to form the catalytic core. The protein factors function to facilitate and stabilize RNA base pairings, and mediate interactions between splice sites.
Our research focuses on the functional roles of the protein splicing factors through identification and characterization of these factors using biochemical methods. We have characterized several proteins and protein complexes involved in various steps of the splicing pathway, including two protein complexes, the NTC required for spliceosome activation, and the NTR that mediates spliceosome disassembly. We have also identified proteins involved in the first catalytic step of the splicing reaction, including Yju2, Cwc22, Cwc24 and Cwc25. The identification of these factors provides further mechanistic insights underlying the catalytic reactions. Through studies of these proteins, we have developed reagents and methods for arresting splicing at different steps of the pathway and for isolation of intermediate complexes. We have found that the spliceosome is highly dynamic in the catalytic phase, and its conformation can readily switch between different states upon change of environmental conditions to direct different chemical reactions, much like self-splicing group II introns. While the pathway is normally guided by DExD/H-box RNA helicases to facilitate the reaction, the purified spliceosome can catalyze different reactions autonomously without the assistance of DExD/H-box proteins under proper ionic conditions. Eight DExD/H-box proteins are involved in the splicing reaction. We are investigating the mechanisms, which underlie their function in mediating spliceosome remodeling and splicing fidelity control.
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