title: 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 (m(2)2G26) 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 m(2)2G26, reversed maf1-antisuppression. A model that emerges is that competition by increased tRNA levels in maf1Δ cells leads to m(2)2G26 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 m(2)2G26 efficiency and reverse antisuppression. Extending this more broadly, we show that a decrease in tRNA synthesis by treatment with rapamycin leads to increased m(2)2G26 modification and that this response is conserved among highly divergent yeasts and human cells.
authors.affiliation: Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Aneeshkumar G
authors.initials: AG
authors.lastname: Arimbasseri
authors.affiliation: Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Nathan H
authors.initials: NH
authors.lastname: Blewett
authors.affiliation: Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: James R
authors.initials: JR
authors.lastname: Iben
authors.affiliation: Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Tek N
authors.initials: TN
authors.lastname: Lamichhane
authors.affiliation: Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Vera
authors.initials: V
authors.lastname: Cherkasova
authors.affiliation: National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland, United States of America.
authors.forename: Markus
authors.initials: M
authors.lastname: Hafner
authors.affiliation: Commissioned Corps, U. S. Public Health Service, Washington, DC, United States of America.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Maraia
mesh_terms: D000595:Amino Acid Sequence; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D057809:HEK293 Cells / Q000187:drug effects; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D012320:RNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000096:biosynthesis / Q000378:metabolism*; D012362:RNA, Transfer, Ser / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000235:genetics / Q000378:metabolism*
chemical_list: C104954:MAF1 protein, S cerevisiae; C000607335:Maf1 protein, S pombe; D012362:RNA, Transfer, Ser; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; D012343:RNA, Transfer; C089041:N(2),N(2)-dimethylguanosine-26-methyltransferase; D012359:tRNA Methyltransferases; D012320:RNA Polymerase III; D020123:Sirolimus