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. 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.

Post-transcriptional modifications of anticodon loop (ACL) nucleotides impact tRNA structure, affinity for the ribosome, and decoding activity, and these activities can be fine-tuned by interactions between nucleobases on either side of the anticodon. A recently discovered ACL modification circuit involving positions 32, 34, and 37 is disrupted by a human disease-associated mutation to the gene encoding a tRNA modification enzyme. We used tRNA-HydroSeq (-HySeq) to examine ³methyl-cytidine-32 (m³C32), which is found in yeast only in the ACLs of tRNAs^Ser and tRNAs^Thr. In contrast to that reported for Saccharomyces cerevisiae in which all m³C32 depends on a single gene, TRM140, the m³C32 of tRNAs^Ser and tRNAs^Thr of the fission yeast S. pombe, are each dependent on one of two related genes, trm140⁺ and trm141⁺, homologs of which are found in higher eukaryotes. Interestingly, mammals and other vertebrates contain a third homolog and also contain m³C at new sites, positions 32 on tRNAs^Arg and C47:3 in the variable arm of tRNAs^Ser. More significantly, by examining S. pombe mutants deficient for other modifications, we found that m³C32 on the three tRNAs^Ser that contain anticodon base A36, requires N⁶-isopentenyl modification of A37 (i⁶A37). This new C32–A37 ACL circuitry indicates that i⁶A37 is a pre- or corequisite for m³C32 on these tRNAs. Examination of the tRNA database suggests that such circuitry may be more expansive than observed here. The results emphasize two contemporary themes, that tRNA modifications are interconnected, and that some specific modifications on tRNAs of the same anticodon identity are species-specific.

In eukaryotes, RNA polymerase (RNAP) III transcribes hundreds of genes for tRNAs and 5S rRNA, among others, which share similar promoters and stable transcription initiation complexes (TIC), which support rapid RNAP III recycling. In contrast, RNAP II transcribes a large number of genes with highly variable promoters and interacting factors, which exert fine regulatory control over TIC lability and modifications of RNAP II at different transitional points in the transcription cycle. We review data that illustrate a relatively smooth continuity of RNAP III initiation-elongation-termination and reinitiation toward its function to produce high levels of tRNAs and other RNAs that support growth and development.