Loss of wobble uridine modification in tRNA anticodons interferes with TOR pathway signaling

Abstract

Previous work in yeast has suggested that modification of tRNAs, in particular uridine bases in the anticodon wobble position (U34), is linked to TOR (target of rapamycin) signaling. Hence, U34 modification mutants were found to be hypersensitive to TOR inhibition by rapamycin. To study whether this involves inappropriate TOR signaling, we examined interaction between mutations in TOR pathway genes (tip41∆, sap190∆, ppm1∆, rrd1∆) and U34 modification defects (elp3∆, kti12∆, urm1∆, ncs2∆) and found the rapamycin hypersensitivity in the latter is epistatic to drug resistance of the former. Epistasis, however, is abolished in tandem with a gln3∆ deletion, which inactivates transcription factor Gln3 required for TOR-sensitive activation of NCR (nitrogen catabolite repression) genes. In line with nuclear import of Gln3 being under control of TOR and dephosphorylation by the Sit4 phosphatase, we identify novel TOR-sensitive sit4 mutations that confer rapamycin resistance and importantly, mislocalise Gln3 when TOR is inhibited. This is similar to gln3∆ cells, which abolish the rapamycin hypersensitivity of U34 modification mutants, and suggests TOR deregulation due to tRNA undermodification operates through Gln3. In line with this, loss of U34 modifications (elp3∆, urm1∆) enhances nuclear import of and NCR gene activation (MEP2, GAP1) by Gln3 when TOR activity is low. Strikingly, this stimulatory effect onto Gln3 is suppressed by overexpression of tRNAs that usually carry the U34 modifications. Collectively, our data suggest that proper TOR signaling requires intact tRNA modifications and that loss of U34 modifications impinges on the TOR-sensitive NCR branch via Gln3 misregulation.

The Levels of a Universally Conserved tRNA Modification Regulate Cell Growth

“…Methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U) is a modification present at position 34 and is required for the efficient decoding by tRNA Lys UUU (10). The absence of mcm 5 s 2 U mislocalizes Gln3p due to reductions in TOR activity (65). Together with our results, these observations suggest that TOR activity is probably affected by the aberrant translation caused by deficiencies in either modification.…”

“…Recently, Scheidt et al (65) showed in yeast that absence of another tRNA modification altered TOR activity as well. Methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U) is a modification present at position 34 and is required for the efficient decoding by tRNA Lys UUU (10).…”

Role of Pseudouridine Formation by Deg1 for Functionality of Two Glutamine Isoacceptor tRNAs

“…In support of a specific requirement for ψ38 in tRNA Gln UUG in the event of wobble base hypomodification, strong synthetic growth defects were observed upon combining the deg1 mutation with mutations in Elongator or Urm1 pathway genes, indicative of functional crosstalk between the modifications at position 34 and 38 in tRNA Gln UUG [8,9,38]. Elongator and Urm1 pathway mutants are further known to exhibit shared phenotypes, including translational inaccuracy and sensitivity to TORC1 inhibiting agents such as caffeine or rapamycin and these are synergistically increased in double mutants lacking both modification activities [9,30,31,32].…”

Dimensionality reduction by UMAP to visualize physical and genetic interactions

“…its absence leads to tRNA modification defects. 16 To assess whether UMAP distance captured known interactions as well as pairwise correlation, we used a dataset of 1060 protein interactions determined from coimmunoprecipitation followed by mass spectrometry. 2 The UMAP clustering data captured these complexes more sensitively and with more precision than previous pairwise correlation-based metrics (AUC pairwise correlation = 0.73, AUC UMAP = 0.84, Figure 2A).…”