Structure-Function Analysis And Genetic Interactions Of Luc7 And Mud1 Subunits Of Saccharomyces Cerevisiae U1 Small Nuclear Ribonucleoprotein
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The discovery of the spliceosome, a large molecular complex composed of five small nuclear ribonucleoprotein particles (snRNPs) functioning in the removal of introns from precursor messenger RNAs stimulated an extensive field of research with innumerable questions on the mechanistic role of each component of the spliceosome. U1 snRNP plays a crucial role in 5' splice site selection and initiates spliceosome assembly for pre-mRNA splicing in S. cerevisiae. The composition of U1 snRNP in S. cerevisiae is much more complex than in humans. This thesis focusses on establishing structure-function relations for Luc7, a 261 amino acid essential subunit of U1 snRNP, by conducting an in vivo mutational analysis entailing N- and C-terminal truncations and alanine scanning of phylogenetically conserved amino acids, including two putative zinc-finger motifs, ZnF1 and ZnF2, and charged amino acids within ZnF2 module. I identified Luc7-(31-246) as a minimal functional protein. Moreover, mutations in the ZnF2 module were found to be lethal which is now explained from structural insights to be due to the H-bonds formed by amino acids in this zinc-finger domain with U1 snRNA: pre-mRNA duplex. I also determined functional roles of otherwise benign mutations and deletions where they impaired splicing of a pre-mRNA with a nonconsensus splice site and were synthetically lethal with other splicing factors. Mud1 is an inessential 298-amino acid subunit of the S cerevisiae U1 snRNP. Mud1 consists of N-terminal and C-terminal RRM domains (RRM1 and RRM2) separated by a linker domain. This thesis dissects genetically the domain requirements of Mud1 by analyzing synthetic lethal interactions of mud1Δ with deletions or mutations in other spliceosome components. Interestingly, I found that all biological activities of Mud1 can be complemented by co-expressing separately the RRM1 (aa 1-127) and linker-RRM2 (aa 128-298) modules. I also investigated the probable role of the linker region and determined it to be a nuclear localizing signal. Thus, the findings in this thesis highlight the contributions of Luc7 and Mud1 in stabilizing the interactions of U1 snRNP with the pre-mRNA 5' splice site.
Genetic screen; RNA; Splicing; Synthetic lethal; U1 snRNP
Doctor of Philosophy
Attribution-NonCommercial-NoDerivatives 4.0 International
dissertation or thesis
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