Project description:
Although pre-mRNA splicing is an essential step in eukaryotic gene expression, the mechanism of the spliceosome function, the multicomponent enzyme responsible for its catalysis, is still poorly understood. We now plan to focus on the role of U6 snRNA, an essential component of the catalytic center of the spliceosome that coordinates metal ions at the active site. U6 has to undergo conformational changes to adopt the catalytically-competent structure, yet we do not know either what is this structure, or how is it stabilized at the catalytic center. We have recently identified a class of C-rich exon sequences similar to U6-ISL sequence that alter splicing, presumably by competing with U6 in its interactions at the catalytic center.
The goal of the current project is to better understand these interactions and the role of C-rich exon motifs in modulation of U6 functions. We will focus on three aims:
1. The structure-function analysis of Cwc2-U6 interactions – the importance of the U6-ISL tetraloop:Cwc2-RNP interaction; we will verify the hypothesis that Cwc2 stabilizes the ‘closed’, catalytic conformation of U6 through its contact with U6-ISL. We will select U6 and cwc2 mutants that alter stability of this interaction and analyze their genetic interactions with alleles of factors that modulate spliceosome transitions in the catalytic phase. 2. Identification of spliceosomal factors that influence function of C-rich, U6-like exons: we will select for mutants that restore the functionality of suboptimal C-rich exons and characterize interactions between exon motifs and the components of the catalytic center, focusing on U6 and Cwc2. 3. Characterization of physical interactions involving C-rich exons within the spliceosome: we will identify spliceosomal components that interact directly with C-rich exon motifs and study the effect of these sequences on spliceosome assembly and splicing in vitro.
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