Functional Analysis of RNA Exosome Mutants Linked to Disease Using a Budding Yeast Model Restricted; Files Only

Enyenihi, Liz (Spring 2020)

Permanent URL: https://etd.library.emory.edu/concern/etds/kk91fm70g?locale=en
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Abstract

Regulation of gene expression at many levels is critical to control the fate and function of all cell types. While transcriptional regulation is important, RNA processing enzymes also play a crucial role in regulating gene expression. The RNA exosome is an evolutionarily conserved, multi-subunit complex that both processes and degrades many classes of RNA. Mutations in genes encoding subunits of the RNA exosome cause a number of significant neurological diseases that lead to drastically shortened lifespans. My research investigates how amino acid substitutions in a structural subunit of the RNA exosome, EXOSC2, affect overall RNA exosome function, with a particular focus on the amino acid substitutions that have been linked to a novel neurodegenerative syndrome in humans. As part of a broader analysis of RNA exosome models that I contributed to, I generated a budding yeast model to assess the functional consequences of missense mutations in EXOSC2 that are linked to disease. Using this model, I conducted experiments to assess the growth, steady state RNA levels, protein levels, and potential cofactor interactions in budding yeast. I found that the yeast ortholog of the EXOSC2 G30V variant was lethal while the yeast ortholog of the EXOSC2 G198D variant caused a temperature sensitive growth defect, reduction in protein level, accumulation of some known RNA exosome substrate transcripts. Furthermore, I exploited yeast genetics approaches to identify genetic interactions with the nonessential RNA exosome cofactors Mpp6 and Rrp47. Taken together, my studies provide insight into how specific amino acid changes could impact RNA exosome function and contribute to disease pathology.

Table of Contents

INTRODUCTION ............................................................................................................. 1

MATERIALS AND METHODS ........................................................................................ 5

S. cerevisiae strains, plasmids, and chemicals ............................................................ 5

S. cerevisiae transformation......................................................................................... 5

S. cerevisiae growth assays......................................................................................... 5

Immunoblotting ............................................................................................................ 6

Total RNA isolation ...................................................................................................... 7

Quantitative RT-PCR ................................................................................................... 8

RESULTS ....................................................................................................................... 9

Novel syndrome patient mutations alter conserved EXOSC2 residues ....................... 9

Amino acid substitutions in S. cerevisiae Rrp4 corresponding to SHRF-associated EXOSC2 substitutions impair growth ......................................................................... 10

Rrp4 protein variants are expressed at similar levels to wildtype Rrp4 ...................... 11

RNA Exosome target transcripts are elevated in rrp4-G226D mutant cells ............... 12

The rrp4-G226D mutant shows genetic interactions with RNA exosome cofactors ... 12

Expression of Heat shock protein 82 does not rescue rrp4-G226D growth defect ..... 13

DISCUSSION ................................................................................................................ 14

REFERENCES .............................................................................................................. 19

FIGURES ...................................................................................................................... 23

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