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

Viktor Scheidt,Raffael Schaffrath,Christian Baer,Andre Juedes,Roland Klassen

doi:10.15698/mic2014.12.179

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.

Highlights

While cell growth and proliferation are typically characterized by active de novo protein synthesis, cell quiescence goes along with translational downregulation
Previously, it was shown that inactivation of either Sit4 or all four Sap proteins required for Sit4 function causes rapamycin hypersensitivity and that this trait is not altered in combination with an rrd1∆ null-allele [17, 23]
This is similar to Sit4 dependent Elongator dephosphorylation, which requires Sap185 and Sap190 and promotes Elongator’s tRNA modification function [25, 27, 28]

Summary

Introduction

While cell growth and proliferation are typically characterized by active de novo protein synthesis, cell quiescence goes along with translational downregulation. This Sit4 role is independent of Rrd1/2 but requires the Sit4 partner proteins Sap185 and Sap190 [17, 23, 29], which is why a sap185∆190∆ double mutant copies defects typical of sit4∆ and Elongator mutants (i.e. Elongator hyperphosphorylation, loss of tRNA modification, zymocin resistance and rapamycin hypersensitivity) [25,26,27,28].

Results

Conclusion