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Title page for ETD etd-09042013-155103
|Type of Document
||Livesay, Stephen Brent
||Spliceosomal GTPase Cwf10: Roles in Splicing in vivo and Characterization of the N-terminal Extension (NTE) Domain
||Cell and Developmental Biology
|Susan R. Wente
- intrinsically disordered
- U5 snRNP
|Date of Defense
The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA, yielding mature message. Schizosaccharomyces pombe Cwf10 (homolog of Saccharomyces cerevisiae Snu114 and human U5-116K), an integral member of the U5 snRNP, is a GTPase that has multiple roles within the splicing cycle.
Cwf10/Snu114 family members are highly homologous to eukaryotic translation elongation factor EF2, and they contain a conserved N-terminal extension (NTE) to the EF2-like portion, predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing. Genetic interactions between cwf10-ΔNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation and second-step catalysis. Characterization of Cwf10-NTE by various biophysical techniques shows that in solution the NTE contains regions of both structure and disorder. When the entire NTE is overexpressed in the cwf10-ΔNTE background, it can complement the truncated Cwf10 protein in trans, and it immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of independently incorporating in to the spliceosome and improving splicing function, possibly indicating a role for the NTE in stabilizing conformational rearrangements during a splice cycle.
A PCR-based random mutagenesis strategy of the cwf10 gene led to the recovery of nine temperature sensitive alleles, all with unique mutations. The mutants displayed phenotypes including splicing defects, heterochromatin-silencing defects, reduced levels of spliceosome proteins, and altered sedimentation of snRNAs in density gradients. Low levels of Cwf10 protein were observed in every mutant, suggesting that the mutations destabilize the protein. We propose that the observed phenotypes are likely caused, in part, by low Cwf10 protein levels.
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