EXPLORING THE CATALYTIC ACTIVITY OF THE [BIG+] PRION AND DYNAMICS OF TRNA T-LOOP MODIFICATIONS IN SACCHAROMYCES CEREVISIAE
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Date
2024-08-07
Authors
Shaw, Ethan
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Publisher
University of Oregon
Abstract
RNA modifications affect stability, structure, and interactions with other molecules. They are important for the optimal functioning of RNA, and without them RNA can be degraded more easily and translation can become less efficient. The majority of known chemical modifications on RNA have been found on tRNAs. The modifications are so tightly packed together that many methods used to detect modifications on other RNAs are ineffective on tRNAs. In this work, I used a new and developing technology to detect RNA modifications - nanopore sequencing. Using direct RNA sequencing of tRNAs to detect modifications, I investigated 1) the catalytic activity of the yeast prion [BIG+] and 2) the incorporation of m5U54, Ψ55, and m1A58 in the T-loop of tRNAs. [BIG+] causes yeast cells to proliferate more quickly with a shorter lifespan allowing them to thrive in nutritionally rich environments. These phenotypes could be caused by altered translation in these cells. Specifically, global translation is increased and certain genes are translated more quickly. However, the mechanism behind these changes is poorly understood. Here, I explore the catalytic activity of [BIG+]. [BIG+] arises from the protein Pus4, a pseudouridine synthase. I hypothesized that a change in the levels of pseudouridylation in [BIG+] cells could be driving the changes in translation. I observed that [BIG+] has retained its catalytic activity and a change in pseudouridylation is unlikely to explain why cells with [BIG+] proliferate more quickly.
Additionally, I co-developed one of the first direct RNA-sequencing methods to comprehensively detect Ψ55 and m1A58 in the T-loop of tRNAs by combining nanopore sequencing of yeast mutants with mass spectrometry. Using this technique, we were the first to validate the presence of a tRNA modification circuit, where Ψ55 promotes the formation of m1A58, in all yeast tRNA isoacceptors. Furthermore, we showed that m1A58 is a dynamic modification that can change under stress. This method is a first step in being able to comprehensively detect and eventually quantify modifications in tRNAs. Being able to accurately detect these modifications will help us understand their complex roles.
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Keywords
Nanopore Sequencing, Prion, RNA modifications, tRNA