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Author

Hema Pingali

Date of Award

2017

Document Type

Restricted Thesis: Campus only access

Degree Name

Bachelor of Science

Department

Biology

Abstract

The nuclear exosome is responsible for processing and degrading various RNAs within the yeast nucleus. Mtr4 is an essential RNA helicase that associates with the nuclear exosome and assists it in RNA processing and degradation. Although the structure of Mtr4 has been solved, the exact mechanism by which Mtr4 aids the nuclear exosome in selectively processing or degrading RNA is still unclear, as are the exact RNA targets of Mtr4 function. To investigate the function of MTR4, I used error-prone PCR to generate a library of mtr4 mutants and established a genetic screen to identify conditional mtr4 mutants. Conditional mutants are fully functional at standard temperatures, but are defective at extreme temperatures. These mutants are useful because their defects can be turned on at command, so the defective proteins can be studied without fear that observed results are related to build up of defect-associated side effects. I completed a pilot screen and am currently completing a larger-scale screen for conditional mutants, implementing lessons learned from the pilot screen. Thus far, I have identified two strongly temperature-sensitive mutants, four subtly temperature-sensitive mutants, and one cold- sensitive mutant from the second round of mutagenesis. Several of these mutants have been sequenced, revealing putative novel conditional mutations. The ability of these conditional mutants to process and degrade RNA can next be assessed by performing a number of in vivo and enzymatic activity assays. The purpose of these studies is to identify individual residues involved in specific functions of Mtr4 to ultimately learn how Mtr4 is able to modulate its function between the two radically different jobs of processing and degrading RNA. These mutants can be used to explore the function and substrates of Mtr4 in vivo and in vitro.

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