Author ORCID Identifier
https://orcid.org/0000-0002-3177-5242
Date of Graduation
12-2022
Document Type
Dissertation (PhD)
Program Affiliation
Experimental Therapeutics
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Ambro van Hoof, PhD
Committee Member
Danielle Garsin, PhD
Committee Member
Kevin Morano, PhD
Committee Member
Michelle Steiger, PhD
Committee Member
Wenbo Li, PhD
Committee Member
William Plunkett, PhD
Abstract
The RNA exosome complex is known to process and/or degrade many classes of RNA, including mRNA, rRNA, tRNA and snRNA, through its 3’-to-5’ exoribonuclease catalytic activity. The RNA exosome complex comprises a 9-unit core (exo-9), with six proteins making up a PH-Ring barrel and three proteins making up a S1-KH cap. The RNA exosome complex exo-9 core is inactive by itself and requires different cofactors in the nucleus and cytoplasm to perform different localized functions. This exo-9 core can associate with one or two catalytic subunits, including Rrp44/Dis3 and Rrp6 in yeast and DIS3 or DIS3L and RRP6/EXOSC10 in humans. The RNA exosome complex plays an essential role in RNA processing and degradation, which is vital for homeostatic gene expression. Mutations that affect RNA exosome complex function lead to aberrant expression of RNAs and, consequently, a variety of maladies.
Because the RNA exosome complex is similar in yeasts and humans in structure and function, I use yeast as a model organism. In yeast, the cytoplasmic RNA exosome complex, which functions to degrade mRNA, binds Ski7 and the SKI complex, which comprises Ski3, Ski8 and the cytoplasmic RNA helicase, Ski2. In the nuclear RNA exosome complex Rrp6 has both catalytic and structural roles in that it mediates the interaction of the RNA exosome complex to other cofactors, such as Rrp47 and the nuclear RNA helicase, Mtr4. Physicians have identified mutations in the exo-9 core in pontocerebellar hypoplasia patients (PCH), in SKIC2 and SKIC3 (the orthologs of Ski2 and Ski3) in tricho-hepato-enteric syndrome (THES) patients, and in Rrp6 in a patient with primary ovarian insufficiency (POI). I hypothesize that these mutations in the RNA exosome complex or cofactors cause different defects in specific RNA exosome complex functions, which result in these diverse disease states.
Although mutations in two SKI complex subunits cause THES, it is not clear from the human patient variant that a complete loss of SKI complex function causes this disease. I showed that causative THES patient mutations modeled in yeast cause severe defects in the cytoplasmic mRNA degradation function of the SKI complex and the RNA exosome complex. Separately, Rrp6 deletion has previously been reported to cause sporulation defects and the report of a human EXOSC10 variant in POI led me to test the function of Rrp6 in sporulation/ gametogenesis. In contrast to what was previously reported, I found that loss of Rrp6 did not have a general sporulation defect. Finally, I investigated Mtr4 to determine whether different mutations can cause different defects. Mtr4 contains a short unstructured N-terminus that is known to interact with Rrp6. I identified three lysine residues in this region that provide Mtr4 with a novel Rrp6-independent function. Overall, these results extend the use of yeast as a model to understand how specific amino acid changes in the RNA exosome complex machinery affect RNA processing.
Keywords
RNA exosome complex, Mtr4, Dob1, Rrp6, EXOSC10, Ski2, SKIC2