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

Arimbasseri AG, Blewett NH, Iben JR, Lamichhane TN, Cherkasova V, Hafner M, Maraia RJ - PLoS Genet. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

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Maf1 is a rapamycin-sensitive regulator of RNAP III-mediated tRNA transcription in S. pombe.A) Northern blot probed for maf1+ mRNA in parent wild type (WT) strain, maf1Δ strain and maf1Δ in which maf1+ is over expressed from a multicopy plasmid. The rpl8+ mRNA serves as a loading control. Each sample was loaded in duplicate at 2X and 1X. B) Northern blot analysis of tRNASerGCU, tRNAAlaUGC and U5 snRNA loading control on the same blot from the three strains indicated above the lanes. C) Quantitation of the tRNAAlaUGC (white bar) and tRNASerGCU (grey) transcripts from three northern blots, including from panel B, using U5 snRNA on the same blots for calibration. Error bars indicate standard deviations of three experiments. D) Spot assay showing growth of S. pombe strains in minimal media (EMM) with or without rapamycin at 100 ng/ml. E) Northern blot comparing the tRNA transcripts indicated in WT and maf1Δ cells one hour after the addition of rapamycin or DMSO carrier alone to the liquid culture media.
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pgen.1005671.g001: Maf1 is a rapamycin-sensitive regulator of RNAP III-mediated tRNA transcription in S. pombe.A) Northern blot probed for maf1+ mRNA in parent wild type (WT) strain, maf1Δ strain and maf1Δ in which maf1+ is over expressed from a multicopy plasmid. The rpl8+ mRNA serves as a loading control. Each sample was loaded in duplicate at 2X and 1X. B) Northern blot analysis of tRNASerGCU, tRNAAlaUGC and U5 snRNA loading control on the same blot from the three strains indicated above the lanes. C) Quantitation of the tRNAAlaUGC (white bar) and tRNASerGCU (grey) transcripts from three northern blots, including from panel B, using U5 snRNA on the same blots for calibration. Error bars indicate standard deviations of three experiments. D) Spot assay showing growth of S. pombe strains in minimal media (EMM) with or without rapamycin at 100 ng/ml. E) Northern blot comparing the tRNA transcripts indicated in WT and maf1Δ cells one hour after the addition of rapamycin or DMSO carrier alone to the liquid culture media.

Mentions: Unlike for all other species examined, S. pombe is naturally resistant to the growth inhibitory effect of rapamycin [26]. Thus, it was important to determine if maf1+ regulates tRNA production in S. pombe and if it does so under TOR control. We created a maf1-deletion strain and showed that it lacked maf1+ mRNA relative to wild type (WT; Fig 1A, WT/vector vs. maf1Δ/vector). Ectopic over-expression of plasmid-borne maf1+ in maf1Δ increased maf1+ mRNA about 4-fold relative to endogenous maf1+ in WT cells (Fig 1A). Levels of tRNAAlaUGC and tRNASerGCU were increased in maf1Δ relative to WT but decreased relative to WT when maf1+ was over expressed (Fig 1B). Quantitation of these tRNAs relative to U5 snRNA, a RNAP II transcript on the same blot, from triplicate cultures on triplicate blots revealed that three levels of maf1+ expression led to three levels of tRNA expression (Fig 1C).


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

Arimbasseri AG, Blewett NH, Iben JR, Lamichhane TN, Cherkasova V, Hafner M, Maraia RJ - PLoS Genet. (2015)

Maf1 is a rapamycin-sensitive regulator of RNAP III-mediated tRNA transcription in S. pombe.A) Northern blot probed for maf1+ mRNA in parent wild type (WT) strain, maf1Δ strain and maf1Δ in which maf1+ is over expressed from a multicopy plasmid. The rpl8+ mRNA serves as a loading control. Each sample was loaded in duplicate at 2X and 1X. B) Northern blot analysis of tRNASerGCU, tRNAAlaUGC and U5 snRNA loading control on the same blot from the three strains indicated above the lanes. C) Quantitation of the tRNAAlaUGC (white bar) and tRNASerGCU (grey) transcripts from three northern blots, including from panel B, using U5 snRNA on the same blots for calibration. Error bars indicate standard deviations of three experiments. D) Spot assay showing growth of S. pombe strains in minimal media (EMM) with or without rapamycin at 100 ng/ml. E) Northern blot comparing the tRNA transcripts indicated in WT and maf1Δ cells one hour after the addition of rapamycin or DMSO carrier alone to the liquid culture media.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697793&req=5

pgen.1005671.g001: Maf1 is a rapamycin-sensitive regulator of RNAP III-mediated tRNA transcription in S. pombe.A) Northern blot probed for maf1+ mRNA in parent wild type (WT) strain, maf1Δ strain and maf1Δ in which maf1+ is over expressed from a multicopy plasmid. The rpl8+ mRNA serves as a loading control. Each sample was loaded in duplicate at 2X and 1X. B) Northern blot analysis of tRNASerGCU, tRNAAlaUGC and U5 snRNA loading control on the same blot from the three strains indicated above the lanes. C) Quantitation of the tRNAAlaUGC (white bar) and tRNASerGCU (grey) transcripts from three northern blots, including from panel B, using U5 snRNA on the same blots for calibration. Error bars indicate standard deviations of three experiments. D) Spot assay showing growth of S. pombe strains in minimal media (EMM) with or without rapamycin at 100 ng/ml. E) Northern blot comparing the tRNA transcripts indicated in WT and maf1Δ cells one hour after the addition of rapamycin or DMSO carrier alone to the liquid culture media.
Mentions: Unlike for all other species examined, S. pombe is naturally resistant to the growth inhibitory effect of rapamycin [26]. Thus, it was important to determine if maf1+ regulates tRNA production in S. pombe and if it does so under TOR control. We created a maf1-deletion strain and showed that it lacked maf1+ mRNA relative to wild type (WT; Fig 1A, WT/vector vs. maf1Δ/vector). Ectopic over-expression of plasmid-borne maf1+ in maf1Δ increased maf1+ mRNA about 4-fold relative to endogenous maf1+ in WT cells (Fig 1A). Levels of tRNAAlaUGC and tRNASerGCU were increased in maf1Δ relative to WT but decreased relative to WT when maf1+ was over expressed (Fig 1B). Quantitation of these tRNAs relative to U5 snRNA, a RNAP II transcript on the same blot, from triplicate cultures on triplicate blots revealed that three levels of maf1+ expression led to three levels of tRNA expression (Fig 1C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus