Author ORCID Identifier
0000-0003-1243-1492
Date of Graduation
5-2023
Document Type
Dissertation (PhD)
Program Affiliation
Microbiology and Molecular Genetics
Degree Name
Doctor of Philosophy (PhD)
Advisor/Committee Chair
Ambro van Hoof
Committee Member
Kevin Morano
Committee Member
Nicholas DeLay
Committee Member
Nayun Kim
Committee Member
Neal Waxham
Abstract
The tRNA splicing endonuclease (TSEN), has been studied for over three decades for its function in tRNA splicing. However, this enzyme has other functions that are just beginning to be characterized. Mutations in TSEN cause the neuronal disease pontocerebellar hypoplasia (PCH) that is characterized by atrophy of the cerebellum and pons, overall developmental failure, and usually results in death before adolescence. How mutations in TSEN cause these neuronal defects and disease is not understood. In yeast, TSEN has another essential function that is independent of tRNA splicing and is still unknown. In this thesis I strived to understand the other function of the TSEN complex. TSEN has one mRNA target in yeast which led me to the hypothesis that TSEN could cleave other mRNAs. I used Parallel Analysis of RNA Ends (PARE) to identify other mRNA substrates of TSEN. I found TSEN cleaves a subset of mRNAs that encode mitochondrial localized proteins. In vivo and in vitro analysis determine TSEN recognizes an A before its cleavage sites. We identified some sequence and localization requirements for TSEN targets but it is likely other factors play a role in substrate recognition such as structure of the mRNA target. Overall we used PARE to identify a novel endonuclease decay pathway, termed TED, in which TSEN can degrade a select group of mRNAs. Yeast genetic screens were used to complement our RNAseq approach to finding the other essential function of TSEN. A spontaneous suppressor screen identified mutations in Dbr1 as suppressors of only the other essential function a mutant sen2. Because mutations in Dbr1 could only complement a partially functional TSEN complex and the catalytic activity of Dbr1 must be lost for this suppression, we propose that Dbr1 and TSEN complete for a common substrate. Through RNAseq, we discovered that loss of the other essential function of TSEN triggers the Gcn4 response. This response is protective in our sen2 mutant and when Dbr1 is mutated in addition to sen2, the Gcn4 response is reduced as TSEN now has no competition for substrate to perform its essential function. As TSEN is involved in mRNA decay through the TED pathway, I wondered what enzymes could be involved in the degradation of these cleavage products. To investigate this, we used PARE to define targets of the exonuclease Dxo1 and the kinase Trl1. This revealed Dxo1 can “nibble” downstream of endonuclease cleavage and decapping but that its main function is in processing the 25S’ to the 25S rRNA in the cytoplasm. Trl1 can also act downstream of endonuclease decay in the TED pathway by phosphorylating the 5’ end of TSEN cleavage products. Though the other essential function of TSEN remains elusive, this research uncovered the participation of TSEN in mRNA decay and the functions of downstream enzymes as well as the identification of a potential competitor, Dbr1.
Keywords
TSEN, endonuclease, PARE, Dbr1, tRNA processing, yeast screens, PCH