Cellular Surveillance and Degradation of Aberant tRNA

Marquette University, Milwaukee WI

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Abstract

DESCRIPTION (provided by applicant): Transfer RNA serves to interpret the information in messenger RNA into proteins that conduct cellular activities. Remarkably, cellular tRNAs also play a role in priming RNA-directed DNA synthesis of several retroviral genomes, including HIV. Thus, elucidating the mechanisms of tRNA formation and function has implications for physiological and pathological forms of gene expression. Newly synthesized pre-tRNAs undergo extensive processing to become competent for nucleocytoplasmic transport and protein synthesis. Most tRNAs attain a similar tertiary structure that depends on a variety of intramolecular interactions and numerous modified nucleosides. This structure is critical for tRNA stability and function. To preserve the fidelity of protein synthesis, it would be beneficial for the cell to detect and eliminate aberrant tRNAs. A mechanism to degrade defective tRNAs has been described in E. coli, but nothing is known about the removal of aberrant tRNAs from eukaryotic cells. The yeast tRNA 1-methyladenosine (m1A) methyltransferase, is a two-subunit enzyme encoded by TRM6 and TRM61. In trm6 and trm61 mutants, m1A formation is blocked and tRNAi Met levels are diminished, suggesting that m1A is critical for tRNAi Met stability. Investigating the mechanism of instability and the role of m1A in tRNA turnover are the focus of this proposal. To identify cellular strategies for overcoming m1A deficiency, we isolated three suppressor genes (sup1-3) that restore tRNAi Met levels in the mutant trm6-504. From these observations and the identity of two of these genes, we have established that tRNAi Met lacking m1A is recognized adenylated and eliminated by the exosome in the nucleus. Whether all subunits of the exosome function equally in degrading hypomodified tRNAi Met will be assessed and the involvement of accessory components of the exosome will be tested. Our work will focus on uncovering the details of this tRNA degradation mechanism by assigning biochemical activities to proteins identified as integral components of the pathway. We will also ask if degradation is limited to hypomodified tRNAs or if other destabilized tRNAs or RNAs are susceptible to turnover in this pathway. This study will provide insight regarding the importance of mlA in tRNA metabolism and has provided tangible evidence of a novel cellular pathway for the surveillance oftRNA processing and the elimination of aberrant tRNAs from eukaryotic cells.