Fig. 4 The TORC1/TORC2 pathway controls the level of the Elongator-dependent modifications. The TORC1/TORC2 pathway controls the level of the Elongator-dependent.

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Fig. 4 The TORC1/TORC2 pathway controls the level of the Elongator-dependent modifications. The TORC1/TORC2 pathway controls the level of the Elongator-dependent modifications. (A) Left: Hierachical clustering analysis of changes in the relative levels of a set of tRNA ribonucleoside modifications in mutants of genes required for the synthesis of wobble s2 (ctu1) and mcm5 (elp3) uridine moieties. The bottom color bar displays the range of log 2 fold change values. Right: Similar to that in the left, except that all detectable Elongator-dependent modified ribonucleosides are shown and the following conditions were applied: treatment with rapamycin, overexpression of tor1 or tor2, deletion of the trm9 gene encoding the enzyme required for the methylation of the cm5 moiety of the mcm5U and mcm5s2U modifications. (B) Phosphorylation assay of peptides corresponding to the amino acids 107 to 120 of fission yeast Elp4 harboring the indicated mutations or phosphorylation (S118P) using immunoprecipitated Gsk3-flag. (C) Phosphorylation assay of endogenous immunoprecipitated Elp4-TAP protein (wt or S114A as indicated) mixed with immunoprecipitated Gsk3-flag before the kinase assay. The band corresponding to Elp4 (40 kDa) is indicated by an arrow. (D) Western blot controlling the level of Gsk3 or Elp4 from the experiment shown in (C). Half of the immunoprecipitation (IP) products were used. Note that the Elp4 S114A mutant is expressed at higher level (see below). Julie Candiracci et al. Sci Adv 2019;5:eaav0184 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).