Path to destruction: tRNA family-specific turnover pathways regulate tRNA intron degradation
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Date
2022-12
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The Ohio State University
Abstract
My project employing the yeast model organism, Saccharomyces cerevisiae, seeks to identify gene products responsible for the destruction of free introns that are removed from precursor transfer RNAs (pre-tRNAs) upon splicing. tRNAs function to deliver amino acids to the ribosome during protein synthesis. One essential step of pre- tRNA maturation is the removal of introns. Each cell cycle in yeast, more than 600,000 free introns are generated; however, introns are rarely detected in wild-type cells because they are rapidly destroyed after splicing.
Ten of S. cerevisiae's 42 tRNA families are encoded by intron-containing genes, but the mechanism for intron turnover is known for only two of these families. In a previous genome-wide screen completed in the Hopper lab, Rlg1 and Xrn1, a known RNA kinase and a known 5' to 3' exonuclease, were shown to be responsible for the turnover of the tRNAIleUAU and the tRNALeuCAA introns. I have been investigating the mechanism of turnover for the other 8 tRNA introns employing a candidate approach. I identified a set of genes potentially responsible for the turnover of free introns by isolating RNA from mutant yeast strains and probing for the accumulation of introns via Northern Blot analysis. To confirm these results, I repeated these experiments using independently generated yeast mutants. I will attempt to complement the intron accumulation phenotype by transforming mutants with plasmids encoding the wild-type genes. If, upon transformation, the phenotype of the mutant cells harboring the wild-type gene reverts back to the wild-type phenotype, the candidate gene must function in the turnover of that particular intron.
My thesis studies will further investigate three new pathways for intron turnover which I have discovered. First, parallel pathways: the kinase Grc3 appears to function in parallel for the turnover of the tRNALysUUU intron, which is degraded in an Rlg1-dependent mechanism. Grc3 mutants result in the accumulation of the tRNALysUUU intron, indicating a redundant kinase function. Second, competing pathways: tRNATrpCCA introns exist as both linear and circular molecules; the linear introns are degraded by Xrn1; however, cells lacking a functional copy of Rrp4, a known 3' to 5' exonuclease, accumulate the circular form of the tRNATrpCCA intron, suggesting bidirectional degradation by Xrn1 and Rrp4. When Xrn1 and Rrp4 are mutated, free introns are able to circularize without attack by exonucleases, resulting in accumulation. Third, an endonuclease catalyzed pathway: we anticipated that an endonuclease destroys circular tRNATrpCCA introns; however, it has not yet been identified. Unexpectedly, non-functional LAS1 and NOB1 results in the accumulation of the tRNALysUUU intron, thus indicating that the Las1 endonucleases could have a novel function cleaving linear introns. By my candidate approach, I still have not identified all necessary nucleases and RNA kinases. Therefore, an unbiased genome-wide screen will be completed to uncover S. cerevisiae genes functioning in intron turnover.
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Keywords
tRNA processing, intron degradation, RNA biology, molecular genetics