Sunday, May 11, 2025
5/11/2025
tRNA Processing - ABSTRACT tRNAs are highly evolved in all organisms for specific recognition by cognate tRNA synthetases, accurate decoding, efficient use in translation, flexibility, and stability. The ubiquitous tRNA modifications are highly conserved in eukaryotes, and many have crucial roles in the yeast Saccharomyces cerevisiae and in human health. Lack of any of several modifications in the tRNA body (outside the anticodon loop) results in growth defects in S. cerevisiae, and is linked to neurological disorders in humans. We study the rapid tRNA decay (RTD) pathway in S. cerevisiae, which targets a subset of mature tRNAs lacking any of four body modifications, due to exposure of the 5' end to the 5'-3' exonucleases Rat1 and Xrn1. RTD also occurs in fully modified tRNA variants with destabilizing mutations exposing the 5' end, and is inhibited in met22Δ mutants due to accumulation of the Met22 substrate adenosine 3',5' bis-phosphate (pAp), which inhibits Rat1 and Xrn1. As little is known about the biology of body modifications in other eukaryotes, we study the evolutionarily distant yeast Schizosaccharomyces pombe, which diverged from S. cerevisiae ~600 million years ago and has facile genetics and molecular biology. Using genetic selection and whole genome sequencing of multiple suppressors of growth defects of body modification mutants, we found that each of two S. pombe body modification mutants target decay of a subset of the hypomodified tRNAs by part, but not all, of the RTD pathway, establishing conservation of the use of this pathway. We also found new interactions between body modifications, the RTD pathway, the ribosome, and a major conserved stress response pathway (the human integrated stress response pathway) in both S. pombe and S. cerevisiae. Our current study of S. pombe pus1Δ mutants, lacking pseudouridine (Ψ), and S. pombe tan1Δ mutants, lacking 4-acetylcytidine (ac4C12), has also yielded new insights. Preliminary results with the pus1Δ mutant show decay of one specific tRNA by the RTD pathway that is responsible for its growth defect, and an unexpected connection between decay and a major conserved pathway that is also linked to the human PUS1 disease phenotype. Preliminary results with the tan1Δ mutants show decay of two tRNAs by the RTD pathway that is responsible for its growth defect, and an unexpected connection between decay and a conserved nuclease not known to have this role. To follow up we will: 1A) Analyze the biology of S. pombe pus1Δ mutants. 1B) Analyze the biology of S. pombe tan1Δ mutants. Despite the importance of tRNA decay, remarkably little is known about the turnover rates of individual wild type (WT) tRNAs or about cellular factors that influence these rates. In addition, little is known about turnover of tRNA ends, although the 3' CCA ends of tRNAs are known to undergo constant repair to ensure healthy growth, and are the focus of several regulatory pathways. To follow up we will 2) Determine tRNA end repair rates and tRNA turnover rates of individual tRNAs, using metabolic labeling and mass spectrometry.
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