Author ORCID Identifier
0009-0008-5795-0150
Date of Graduation
12-2025
Document Type
Thesis (MS)
Program Affiliation
Genetics and Epigenetics
Degree Name
Masters of Science (MS)
Advisor/Committee Chair
Ambro van Hoof
Committee Member
Michael Lorenz
Committee Member
Catherine Denicourt
Committee Member
George Eisenhoffer
Committee Member
Kuang-Lei Tsai
Abstract
A subset of eukaryotic pre-tRNAs have introns that must be spliced out to become mature molecules capable of functioning in translation. This essential processing step is performed by tRNA splicing endonuclease (TSEN), a highly conserved heterotetramer with two catalytic and two structural subunits. In addition to this conserved function, archaeal TSEN also processes pre-rRNA and yeast TSEN cleaves some mRNAs that encode mitochondrial proteins to initiate mRNA degradation. Interestingly, single amino acid mutations in TSEN cause the human Mendelian disease pontocerebellar hypoplasia (PCH) by an unknown mechanism. Recently, several groups have determined the structure of human TSEN, revealing similarities and differences between the human, yeast, and archaeal versions of the complex. This advance afforded us the opportunity to characterize the in vivo relationship between structure and function in eukaryotic TSEN. With these discoveries informing my approach, I have endeavored to uncover the significance of features unique to eukaryotic TSEN, investigate what parts of the complex are responsible for the mitochondrial localization of eukaryotic TSEN, assess the effects of point mutations to residues involved in cleavage, and identify structural and functional features of TSEN-linked PCH. The model organism S. cerevisiae was used to explore these questions, and several mutant strains were prepared. Each strain was created with the intention to investigate a specific aspect of these questions. In each case, the in vivo effects of the mutations were examined through serial dilution growth assays and protein quantitation through western blotting and normalization of band intensity to the Pgk1 protein. In the course of my work, I have identified new information about how eukaryote-specific residues and domains of multiple TSEN subunits contribute to the proper function of their corresponding protein. I have also found that altering TSEN residues involved in cleavage does not appear to significantly affect protein stability but does have detrimental effects on cell viability. Through genetic manipulation and fluorescence microscopy techniques, we have generated new evidence that a specific region of Sen54 is vital for the mitochondrial localization of TSEN and found intragenic suppressor mutants that limit the adverse effects of deleting this region on the health of the cell. Finally, I have shown that modeling TSEN-linked clinical PCH variants in yeast can have a wide range of effects on cell health and protein stability. In summary, this work provides new insight into the structure and function relationships that govern eukaryotic TSEN’s activity while also opening additional questions about these relationships that deserve further inquiry.
Recommended Citation
Underwood, Justin G., "Structure and Function Studies on tRNA Splicing Endonuclease" (2025). Dissertations & Theses (Open Access). 1491.
https://digitalcommons.library.tmc.edu/utgsbs_dissertations/1491
Keywords
TSEN, tRNA, PCH, yeast, splicing, endonuclease