Degradation of several hypomodified mature tRNA species in Saccharomyces cerevisiae is mediated by Met22, and the 5’-3’ exonucleases Rat1 and Xrn1

Abstract

Thesis (Ph. D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Biochemistry and Biophysics, 2008.tRNAs are extremely stable RNA molecules that are exquisitely formed for their specific roles in translation. Formation of fully functional tRNA in all organisms involves multiple processing steps, including the addition of numerous modifications, 25 of which are found in the yeast Saccharomyces cerevisiae. This work describes a novel tRNA degradation pathway that acts on several mature tRNA species lacking combinations of modifications, underscoring the importance of tRNA modifications for tRNA stability and function in vivo. Recent work in this laboratory showed that tRNAVal(AAC) lacking the m7G46 and m5C49 modifications due to mutation of TRM8 and TRM4 is rapidly degraded at 37 °C by a novel mechanism. It is demonstrated here that loss of tRNAVal(AAC) causes the temperature sensitive defect of trm8-Δ trm4-Δ mutants. Furthermore, degradation of hypomodified tRNAVal(AAC) is accompanied by loss of its aminoacylation and occurs at the level of mature tRNA. Analysis of spontaneous suppressors of the temperature sensitive phenotype of trm8-Δ trm4-Δ mutants demonstrates that this mature tRNA degradation pathway is mediated by Met22, and the 5’-3’ exonucleases Rat1 and Xrn1. Mutation or deletion of MET22 completely rescues the temperature sensitive growth defect of the trm8-Δ trm4-Δ strain, and prevents both degradation and loss of aminoacylation of tRNAVal(AAC). Loss of Met22 function has previously been linked to inhibition of Rat1 and Xrn1 due to accumulation of v the Met22 substrate pAp. Consistent with this, mutation of the essential RAT1 gene together with deletion of XRN1 also completely stabilizes both the levels and aminoacylation of hypomodified tRNAVal(AAC) at 37 °C, and rescues growth of trm8-Δ trm4-Δ mutants. Furthermore, this tRNA degradation pathway acts on multiple mature tRNA species, and might play a wider role in turnover of mature tRNA in vivo. It has recently been shown that levels of tRNASer(CGA) and tRNASer(UGA) lacking Um44 and ac4C12 decrease in a temperature dependent fashion, leading to temperature sensitive growth of the trm44-Δ tan1-Δ strain. It is shown here that deletion of MET22 also prevents degradation of mature hypomodified tRNASer(CGA) and tRNASer(UGA), and that mutation of RAT1 in the trm44-Δ tan1-Δ background suppresses the temperature sensitive defect of this strain