Novel role of aaRS in tRNA biology in S. cerevisiae
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Date
2016-05
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The Ohio State University
Abstract
Aminoacyl tRNA synthetases (aaRS) are enzymes that add particular amino acids to cognate tRNAs. These aaRS are essential for the housekeeping function of protein synthesis. We recently reported that temperature sensitive (ts) mutantions of some of the genes encoding aaRSs are defective in pre-tRNA splicing and accumulate end-processed intron containing tRNAIle UAU in S.cerevisiae at the non-permissive temperature. These previous studies identified 5 mutant aaRS genes: ils1, gln4, ths1, frs2, and cdc60 (Wu et al, 2015). My further analysis showed ala1 also causes accumulation of unspliced tRNAIle UAU, but eleven other ts aaRSs do not. The splicing defect is surprising since aaRSs are not known to affect tRNA processing steps. This gives rise to the idea that aaRSs may possess novel activities in tRNA biosynthesis. I am conducting a systematic analysis utilizing a non-radioactive northern method for all aaRS mutants which
have been isolated, 16 out of the 20 aaRS, to determine which aaRS may affect any of the 10 families of tRNAs that are encoded by intron-containing genes. In addition, I am testing three possible mechanisms by which faulty aaRS would cause defects in pre-tRNA intron removal: defective tRNA nuclear export to the cytoplasm, failure to deliver tRNA to mitochondria where intron splicing takes place in yeast, or defects in the splicing mechanism itself. Studies employing
fluorescent in situ hybridization (FISH) techniques investigated the possibilities by determining the subcellular location of the unspliced tRNAs. They were not located in the nucleus, which leads to the conclusion the defect is most likely not
due to inefficient tRNA nuclear export. This leaves the other alternative two possibilities mentioned above. Completion of the study will offer insight to the tRNA biological pathway and a novel role of the aaRS.
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Keywords
tRNA splicing, tRNA nuclear-cytoplasmic traffic, tRNA aminoacyl syntheses