Initiator tRNA lacking 1-methyladenosine is targeted by the rapid tRNA decay pathway in evolutionarily distant yeast species

Abstract

All tRNAs have numerous modifications, lack of which often results in growth defects in the budding yeast Saccharomyces cerevisiae and neurological or other disorders in humans. In S. cerevisiae, lack of tRNA body modifications can lead to impaired tRNA stability and decay of a subset of the hypomodified tRNAs. Mutants lacking 7-methylguanosine at G46 (m7G46), N2,N2-dimethylguanosine (m2,2G26), or 4-acetylcytidine (ac4C12), in combination with other body modification mutants, target certain mature hypomodified tRNAs to the rapid tRNA decay (RTD) pathway, catalyzed by 5’-3’ exonucleases Xrn1 and Rat1, and regulated by Met22. The RTD pathway is conserved in the phylogenetically distant fission yeast Schizosaccharomyces pombe for mutants lacking m7G46. In contrast, S. cerevisiae trm6/gcd10 mutants with reduced 1-methyladenosine (m1A58) specifically target pre-tRNAiMet(CAU) to the nuclear surveillance pathway for 3’-5’ exonucleolytic decay by the TRAMP complex and nuclear exosome. We show here that the RTD pathway has an unexpected major role in the biology of m1A58 and tRNAiMet(CAU) in both S. pombe and S. cerevisiae. We find that S. pombe trm6Δ mutants lacking m1A58 are temperature sensitive due to decay of tRNAiMet(CAU) by the RTD pathway. Thus, trm6Δ mutants had reduced levels of tRNAiMet(CAU) and not of eight other tested tRNAs, overexpression of tRNAiMet(CAU) restored growth, and spontaneous suppressors that restored tRNAiMet(CAU) levels had mutations in dhp1/RAT1 or tol1/MET22. In addition, deletion of cid14/TRF4 in the nuclear surveillance pathway did not restore growth. Furthermore, re-examination of S. cerevisiae trm6 mutants revealed a major role of the RTD pathway in maintaining tRNAiMet(CAU) levels, in addition to the known role of the nuclear surveillance pathway. These findings provide evidence for the importance of m1A58 in the biology of tRNAiMet(CAU) throughout eukaryotes, and fuel speculation that the RTD pathway has a major role in quality control of body modification mutants throughout fungi and other eukaryotes.

Different modification pathways for m1A58 incorporation in yeast elongator and initiator tRNAs

“…The origin of the essentiality of the m 1 A58 modification has been extensively studied and was shown to be required for the maturation and accumulation of the initiator yeast tRNA i Met (43). Hypomodified initiator tRNA i Met lacking m 1 A58 are targeted by the nuclear surveillance pathway and decayed by the TRAMP complex and the nuclear exosome (26,27) as well as by the RTD pathway (41). Importantly, yeast tRNA i Met has an unusual T-loop sequence, and contains unmodified A54 and U55, which are involved in a particular tRNA elbow structure (42).…”

“…However, the three-dimensional structure of the tRNA is not properly formed as demonstrated by the lack of signals attesting of a properly assembled tRNA elbow structure, namely imino groups of U55 and G18 (Figure 4). These structural rearrangements of yeast tRNAi Met upon m 1 A58 modification are most probably at the origin of the specific degradation of hypomodified tRNAi Met lacking m 1 A58 by the nuclear surveillance and RTD pathways (26,27,40,41), the properly folded m 1 A58-tRNAi Met being protected from degradation. It is noteworthy that other hypomodified tRNAs lacking at least one tRNA core modification are targeted to degradation by the RTD pathway.…”

“…Altogether, these reports led to the model that m 1 A58 is introduced on pre-tRNAi Met initial transcripts, which stabilizes the tRNAi Met unique substructure, thereby preventing its degradation by the nuclear surveillance and RTD pathways. In addition, degradation of tRNAi Met lacking m 1 A58 by the RTD pathway was recently shown to be conserved in the phylogenetically distant yeast species S. pombe and S. cerevisiae (41), suggesting that throughout eukaryotes the m 1 A58 modification is crucial to tRNAi Met biology.…”

“…However, previous reports suggested that m 1 A58 is introduced early along the tRNA modification process in yeast, with m 1 A58 being introduced on initial pre-tRNA transcripts (6). Yeast initiator pre-tRNA i Met lacking m 1 A58, but containing the 5’-leader and part of the 3’-trailer sequences, is indeed targeted by the nuclear surveillance and RTD pathways (26,27,40,41). In yeast tRNA i Met , the m 1 A58 modification is part of an unusual tRNA elbow structure involving non-canonical nucleotides A20, A54 and A60.…”

“…Altogether, these reports led to the model that m 1 A58 is introduced on pre-tRNA i Met initial transcripts, which stabilizes the tRNA i Met unique substructure, thereby preventing its degradation by the nuclear surveillance and RTD pathways. In addition, degradation of tRNA i Met lacking m 1 A58 by the RTD pathway was recently shown to be conserved in the phylogenetically distant yeast species S. pombe and S. cerevisiae (41), suggesting that throughout eukaryotes the m 1 A58 modification is crucial to tRNA i Met biology. Here, aiming to reconcile the apparent inconsistency regarding the incorporation of m 1 A58 in yeast tRNAs, namely as a late modification in elongator tRNA Phe and as an early modification in initiator tRNA i Met , we decided to examine the m 1 A58 modification pathways in yeast elongator and initiator tRNAs.…”