Structure and Function Studies on tRNA Splicing Endonuclease

<p>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 <em>S. cerevisiae</em> 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.</p>

Name thesis:degree_name

Masters of Science (MS)

Level thesis:degree_level

Thesis (MS)

Year dc:date.available

2025

Authors dc:creator

• Underwood, Justin G
• <p>0009-0008-5795-0150</p>

Contributors dc:contributor

• Ambro van Hoof
• Michael Lorenz
• Catherine Denicourt

• TSEN
• tRNA
• PCH
• yeast
• splicing
• endonuclease
• Molecular Genetics
• Other Microbiology

Repository record dc:identifier

https://digitalcommons.library.tmc.edu/utgsbs_dissertations/1491

OAI identifier oai:identifier

oai:digitalcommons.library.tmc.edu:utgsbs_dissertations-2548