tRNA^Glu wobble uridine methylation by Trm9 identifies Elongator's key role for zymocin‐induced cell death in yeast

Abstract

SummaryZymocin-induced cell death in Saccharomyces cerevisiae requires the toxin-target (TOT) effector Elongator, a protein complex with functions in transcription, exocytosis and tRNA modification. In line with the latter, trm9 ∆ ∆ ∆ ∆ cells lacking a tRNA methylase specific for wobble uridine (U 34 ) residues survive zymocin and in excess, the Trm9 substrate tRNA Glu copies zymocin protection of Elongator mutants. Phenotypes typical of a tot3/elp3 ∆ ∆ ∆ ∆ Elongator mutant are absent from trm9 ∆ ∆ ∆ ∆ cells but copied in a tot3 ∆ ∆ ∆ ∆ trm9 ∆ ∆ ∆ ∆ double mutant suggesting that Elongator acts upstream of Trm9. Consistent with Elongator-dependent tRNA modification being more important to mRNA decoding than Trm9, SUP4 and SOE1 tRNA suppressors are highly sensitive to loss of Elongator and tRNA U 34 hypomodification. As Trm9 overexpression counteracts the effect of high-copy tRNA Glu

tRNA modifications: Necessary for correct tRNA‐derived fragments during the recovery from stress?

“…For instance, bacteria specifically cleave their own tRNAs in a suicidal attempt to defend the population against infection by bacteriophages [6]. In another example, the yeast Kluyveromyces lactis employs a ''killer'' endonuclease that attacks the anti-codon loops of a variety of Saccharomyces cerevisiae tRNAs, thereby inhibiting the proliferation of a non-self yeast species [16]. In both cases, a modified uridine (5-methoxycarbonyl-methyl-2-thiouridine (mcm5s2U)) in the wobble position (U34) of the anticodon of the targeted tRNAs is essential for endonuclease activity at this site [17] (Fig.…”

Glutaredoxin GRXS17 Associates with the Cytosolic Iron-Sulfur Cluster Assembly Pathway

“…In the light of these findings, it is interesting to note that the GRX3 gene was identified previously as a high-copy suppressor in yeast of growth inhibition by zymocin, a fungal tRNase ribotoxin complex (Jablonowski et al, 2001;Jablonowski and Schaffrath, 2007). Zymocin activity targets elongator-dependent mcm 5 s 2 U34 modifications in tRNA anticodons (Lu et al, 2005;Jablonowski et al, 2006) and eventually kills S. cerevisiae cells. Therefore, loss of tRNA modification in elongator (elp) or tRNA methyltransferase (trm9) mutants protects against zymocin, making the tRNase a useful tool for diagnosing elongator function and, hence, tRNA modification in yeast (Nandakumar et al, 2008) and potentially in Arabidopsis (Mehlgarten et al, 2010;Leitner et al, 2015).…”

“…5A). This phenotype can be suppressed by read-through of SUP4, an ochre suppressor tRNA Tyr carrying a U 34 mutation in its anticodon (Goodman et al, 1977;Huang et al, 2005;Jablonowski et al, 2006; Fig. 5A).…”

“…5A). Because the SUP4 tRNA Tyr needs an mcm 5 -modified U 34 to decode the UAA triplet as Tyr Jablonowski et al, 2006), it cannot suppress the ade2-1ochre reporter without proper mcm 5 formation at U 34 . SUP4 failed to promote stop codon read-through of ade2-1 ochre and hence, conferred adenine auxotrophy in the yeast reporter strain lacking GRX3 function, even if GRX4 was not repressed (Fig.…”

“…Routine yeast growth was performed in rich yeast extract, peptone dextrose (YPD), or Gal (YPG) media or on SD medium containing 2% (w/v) dextrose (SD Glu) or 2% (w/v) Gal (SD Gal;Sherman, 2002). Both lacked adenine to check for ade2-1 ochre read-through by the tRNA suppressor SUP4 and formation of adenine prototrophic cells (Jablonowski et al, 2006). The latter involved growth for 3 d at 30°C of 10-fold serial dilutions of yeast strains W303-1A and pGAL-GRX4/grx3D transformed with SUP4 on a single-copy plasmid pTC3 (Shaw and Olson, 1984;Jablonowski et al, 2009).…”