RNA Polymerase III Output Is Functionally Linked to tRNA Dimethyl-G26 Modification

Abstract

Control of the differential abundance or activity of tRNAs can be important determinants of gene regulation. RNA polymerase (RNAP) III synthesizes all tRNAs in eukaryotes and it derepression is associated with cancer. Maf1 is a conserved general repressor of RNAP III under the control of the target of rapamycin (TOR) that acts to integrate transcriptional output and protein synthetic demand toward metabolic economy. Studies in budding yeast have indicated that the global tRNA gene activation that occurs with derepression of RNAP III via maf1-deletion is accompanied by a paradoxical loss of tRNA-mediated nonsense suppressor activity, manifested as an antisuppression phenotype, by an unknown mechanism. We show that maf1-antisuppression also occurs in the fission yeast S. pombe amidst general activation of RNAP III. We used tRNA-HydroSeq to document that little changes occurred in the relative levels of different tRNAs in maf1Δ cells. By contrast, the efficiency of N2,N2-dimethyl G26 (m22G26) modification on certain tRNAs was decreased in response to maf1-deletion and associated with antisuppression, and was validated by other methods. Over-expression of Trm1, which produces m22G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m22G26 hypomodification due to limiting Trm1, reducing the activity of suppressor-tRNASerUCA and accounting for antisuppression. Consistent with this, we show that RNAP III mutations associated with hypomyelinating leukodystrophy decrease tRNA transcription, increase m22G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m22G26 modification and that this response is conserved among highly divergent yeasts and human cells. Author Summary: Transfer RNAs (tRNAs) are molecular adapters necessary for translation of the genetic code from DNA to messenger RNA (mRNA) to synthesis of the proteins that constitute the energy producing enzymes and structural components of all cells and organisms on earth. In eukaryotic cells, tRNAs are synthesized by RNA polymerase III (RNAP III). Proteins are composed of different amino acids, sequentially arranged according to the triplet sequence code, each carried to the ribosome by a different tRNA which reads or decodes the triplet sequence of the mRNA. After their synthesis by RNAP III, tRNAs are chemically modified by enzymes on multiple of their nucleosides. Here we report that one of these modifications, dimethyl-guanine-26 (m22G26) varies in the efficiency by which it is added to its target tRNAs, in a manner that is dependent on the overall activity rate of RNAP III. We show that this is important because the m22G26 modification activates the tRNA for function to translate the code. This link between RNAP III activity rate and m22G26 modification efficiency was previously unknown, and we show that it is conserved from yeast to human cells.

Publication information

Originally published in PLOS Genetics on 2015-12-31. Original DOI: 10.1371/journal.pgen.1005671.

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