Methodology for the High-Throughput Identification and Characterization of tRNA Variants That Are Substrates for a tRNA Decay Pathway

Payea, Matthew J.; Guy, Michael P.; Phizicky, Eric M.

doi:10.1016/bs.mie.2015.03.003

Cited by 5 publications

(5 citation statements)

References 22 publications

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“…To directly determine if the similar in vivo tRNA decay ratios of anticodon stem variants and acceptor stem variants were due to similar 5 ′ end accessibility, we measured 5 ′ -3 ′ exonuclease activity in vitro on a set of SUP4 οc variants, as we had previously done with tS(CGA) variants and Xrn1 (Whipple et al 2011). To assay decay, we used a biotinylated oligomer to purify SUP4 οc variants together with endogenous tY(GUA), incubated the tRNAs with purified preparations of Xrn1 or Rat1/Rai1 complex (Xue et al 2000;Xiang et al 2009), and assayed decay by poison primer extension, using the endogenous purified tY (GUA) as a control (Payea et al 2015).…”

Section: Resultsmentioning

confidence: 99%

Mutations in the anticodon stem of tRNA cause accumulation and Met22-dependent decay of pre-tRNA in yeast

Payea

Hauke

Zoysa

et al. 2019

RNA

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During tRNA maturation in yeast, aberrant pre-tRNAs are targeted for 3 ′ ′ ′ ′ ′ -5 ′ ′ ′ ′ ′ degradation by the nuclear surveillance pathway, and aberrant mature tRNAs are targeted for 5 ′ ′ ′ ′ ′ -3 ′ ′ ′ ′ ′ degradation by the rapid tRNA decay (RTD) pathway. RTD is catalyzed by the 5 ′ ′ ′ ′ ′ -3 ′ ′ ′ ′ ′ exonucleases Xrn1 and Rat1, which act on tRNAs with an exposed 5 ′ ′ ′ ′ ′ end due to the lack of certain body modifications or the presence of destabilizing mutations in the acceptor stem, T-stem, or tRNA fold. RTD is inhibited by mutation of MET22, likely due to accumulation of the Met22 substrate adenosine 3 ′ ′ ′ ′ ′ ,5 ′ ′ ′ ′ ′ bis-phosphate, which inhibits 5 ′ ′ ′ ′ ′ -3 ′ ′ ′ ′ ′ exonucleases. Here we provide evidence for a new tRNA quality control pathway in which intron-containing pre-tRNAs with destabilizing mutations in the anticodon stem are targeted for Met22-dependent pre-tRNA decay (MPD). Multiple SUP4 οc anticodon stem variants that are subject to MPD each perturb the bulge-helix-bulge structure formed by the anticodon stem-loop and intron, which is important for splicing, resulting in substantial accumulation of end-matured unspliced pre-tRNA as well as pre-tRNA decay. Mutations that restore exon-intron structure commensurately reduce pre-tRNA accumulation and MPD. The MPD pathway can contribute substantially to decay of anticodon stem variants, since pre-tRNA decay is largely suppressed by removal of the intron or by restoration of exon-intron structure, each also resulting in increased tRNA levels. The MPD pathway is general as it extends to variants of tRNA Tyr(GUA) and tRNA Ser(CGA) . These results demonstrate that the integrity of the anticodon stem-loop and the efficiency of tRNA splicing are monitored by a quality control pathway.

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Section: Resultsmentioning

confidence: 99%

Mutations in the anticodon stem of tRNA cause accumulation and Met22-dependent decay of pre-tRNA in yeast

Payea

Hauke

Zoysa

et al. 2019

RNA

Self Cite

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“…Met22 has also been implicated in surveillance of pre-tRNA containing mutations that affect the structure of the intron-exon junction of any of several pre-tRNAs through Met22-dependent pre-tRNA decay (MPD), leading to accumulation of end matured, intron-containing pre-tRNA (Payea et al 2015). Although deletion of trm10 or growth in 5FU also causes accumulation of pre-tRNAs (Figure 3) , no further accumulation of pre-tRNAs was observed in a met22Δ -dependent manner that would suggest met22Δ is similarly acting to stabilize pre- tRNA Trp .…”

Section: Discussionmentioning

confidence: 99%

A tRNA-specific function for tRNA methyltransferase Trm10 is associated with a new tRNA quality control mechanism inSaccharomyces cerevisiae

Bowles,

Jackman

2023

Preprint

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InSaccharomyces cerevisiaea single homolog of the tRNA methyltransferase Trm10 performs m1G9 modification on 13 different tRNAs. Here we provide evidence that the m1G9 modification catalyzed byS. cerevisiaeTrm10 plays a biologically important role for one of these tRNA substrates, tRNATrp. Overexpression of tRNATrp(and not any of 38 other elongator tRNAs) rescues growth hypersensitivity of thetrm10Δstrain in the presence of the antitumor drug 5-fluorouracil (5FU). Mature tRNATrpis depleted intrm10Δcells, and its levels are further decreased upon growth in 5FU, while another Trm10 substrate (tRNAGly) is not affected under these conditions. Thus, m1G9 inS. cerevisiaeis another example of a tRNA modification that is present on multiple tRNAs but is only essential for the biological function of one of those species. In addition to the effects of m1G9 on mature tRNATrp, precursor tRNATrpspecies accumulate in the same strains, an effect that is due to at least two distinct mechanisms. The levels of mature tRNATrpare rescued in thetrm10Δmet22Δstrain, consistent with the known role of Met22 in tRNA quality control, where deletion ofmet22causes inhibition of 5’-3’ exonucleases that catalyze tRNA decay. However, none of the known Met22-associated exonucleases appear to be responsible for decay of hypomodified tRNATrp, based on inability of mutants of each enzyme to rescue growth of thetrm10Δstrain in the presence of 5FU. Thus, the surveillance of tRNATrpappears to constitute a distinct tRNA quality control pathway inS. cerevisiae.

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“…Met22 has also been implicated in surveillance of pre-tRNA containing mutations that affect the structure of the intron-exon junction of any of several pre-tRNAs through Met22dependent pre-tRNA decay (MPD), leading to accumulation of end matured, intron-containing pre-tRNA (Payea et al 2015). Although deletion of trm10 or growth in 5FU also causes accumulation of pre-tRNAs (Figure 3), no further accumulation of pre-tRNAs was observed in a met22Δ-dependent manner that would suggest met22Δ is similarly acting to stabilize pre-tRNA Trp .…”

Section: Discussionmentioning

confidence: 99%

A tRNA-specific function for tRNA methyltransferase Trm10 is associated with a new tRNA quality control mechanism inSaccharomyces cerevisiae

Bowles,

Jackman

2023

RNA

View full text Add to dashboard Cite

In Saccharomyces cerevisiae a single homolog of the tRNA methyltransferase Trm10 performs m1G9 modification on 13 different tRNAs. Here we provide evidence that the m1G9 modification catalyzed by S. cerevisiae Trm10 plays a biologically important role for one of these tRNA substrates, tRNATrp. Overexpression of tRNATrp (and not any of 38 other elongator tRNAs) rescues growth hypersensitivity of the trm10Δ strain in the presence of the antitumor drug 5-fluorouracil (5FU). Mature tRNATrp is depleted in trm10Δ cells, and its levels are further decreased upon growth in 5FU, while another Trm10 substrate (tRNAGly) is not affected under these conditions. Thus, m1G9 in S. cerevisiae is another example of a tRNA modification that is present on multiple tRNAs but is only essential for the biological function of one of those species. In addition to the effects of m1G9 on mature tRNATrp, precursor tRNATrp species accumulate in the same strains, an effect that is due to at least two distinct mechanisms. The levels of mature tRNATrp are rescued in the trm10Δmet22Δ strain, consistent with the known role of Met22 in tRNA quality control, where deletion of met22 causes inhibition of 5'-3' exonucleases that catalyze tRNA decay. However, none of the known Met22-associated exonucleases appear to be responsible for decay of hypomodified tRNATrp, based on inability of mutants of each enzyme to rescue growth of the trm10Δ strain in the presence of 5FU. Thus, the surveillance of tRNATrp appears to constitute a distinct tRNA quality control pathway in S. cerevisiae.

show abstract

Methodology for the High-Throughput Identification and Characterization of tRNA Variants That Are Substrates for a tRNA Decay Pathway

Cited by 5 publications

References 22 publications

Mutations in the anticodon stem of tRNA cause accumulation and Met22-dependent decay of pre-tRNA in yeast

Mutations in the anticodon stem of tRNA cause accumulation and Met22-dependent decay of pre-tRNA in yeast

A tRNA-specific function for tRNA methyltransferase Trm10 is associated with a new tRNA quality control mechanism inSaccharomyces cerevisiae

A tRNA-specific function for tRNA methyltransferase Trm10 is associated with a new tRNA quality control mechanism inSaccharomyces cerevisiae

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