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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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The m22G26 modification efficiency response is conserved in S. cerevisiae and human cells.A) PHA26 assay of S. cerevisiae maf1Δ and WT (MAF1) cells. B) TMS assay shows that over-expression of TRM1 reverses antisuppression phenotype of S. cerevisiae maf1Δ cells. C) PHA26 assay of human embryonic kidney (HEK) 293 cells grown for a period of serum starvation or after serum replenishment as indicated above the lanes. D) PHA26 assay of HEK293 cells in the presence or absence of rapamycin. Quantitative modification indices are shown for panels A, C and D, described as for Fig 4D.
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pgen.1005671.g007: The m22G26 modification efficiency response is conserved in S. cerevisiae and human cells.A) PHA26 assay of S. cerevisiae maf1Δ and WT (MAF1) cells. B) TMS assay shows that over-expression of TRM1 reverses antisuppression phenotype of S. cerevisiae maf1Δ cells. C) PHA26 assay of human embryonic kidney (HEK) 293 cells grown for a period of serum starvation or after serum replenishment as indicated above the lanes. D) PHA26 assay of HEK293 cells in the presence or absence of rapamycin. Quantitative modification indices are shown for panels A, C and D, described as for Fig 4D.

Mentions: To gain insight into a potential mechanism controlling the differential m22G26 modification levels in minimal and rich media, we examined Trm1 levels in extracts from the WT cells grown in YES and EMM and the trm1+ over-expressing cells in EMM by western blotting using tubulin on the same blot as a loading control (Fig 6D). Surprisingly, this showed similar levels of endogenous Trm1 in extracts from cells in grown in YES and EMM (Fig 7D, lanes 1, 2). The over-expressed 3X-FLAG-tagged Trm1 was observed as a slower migrating band in lanes 3 and 4 indicated to the right of Fig 6D. Quantification using Odyssey infrared imaging revealed that the 3X-FLAG-Trm1 accumulated to about 4-fold higher than endogenous Trm1 in the same cell extracts (lanes 3, 4). We conclude that while m22G26 modification efficiency differs dramatically in EMM and YES this is not reflected by Trm1 polypeptide levels detectable by western blotting, whereas over-expression of Trm1 is readily observed.


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)

The m22G26 modification efficiency response is conserved in S. cerevisiae and human cells.A) PHA26 assay of S. cerevisiae maf1Δ and WT (MAF1) cells. B) TMS assay shows that over-expression of TRM1 reverses antisuppression phenotype of S. cerevisiae maf1Δ cells. C) PHA26 assay of human embryonic kidney (HEK) 293 cells grown for a period of serum starvation or after serum replenishment as indicated above the lanes. D) PHA26 assay of HEK293 cells in the presence or absence of rapamycin. Quantitative modification indices are shown for panels A, C and D, described as for Fig 4D.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005671.g007: The m22G26 modification efficiency response is conserved in S. cerevisiae and human cells.A) PHA26 assay of S. cerevisiae maf1Δ and WT (MAF1) cells. B) TMS assay shows that over-expression of TRM1 reverses antisuppression phenotype of S. cerevisiae maf1Δ cells. C) PHA26 assay of human embryonic kidney (HEK) 293 cells grown for a period of serum starvation or after serum replenishment as indicated above the lanes. D) PHA26 assay of HEK293 cells in the presence or absence of rapamycin. Quantitative modification indices are shown for panels A, C and D, described as for Fig 4D.
Mentions: To gain insight into a potential mechanism controlling the differential m22G26 modification levels in minimal and rich media, we examined Trm1 levels in extracts from the WT cells grown in YES and EMM and the trm1+ over-expressing cells in EMM by western blotting using tubulin on the same blot as a loading control (Fig 6D). Surprisingly, this showed similar levels of endogenous Trm1 in extracts from cells in grown in YES and EMM (Fig 7D, lanes 1, 2). The over-expressed 3X-FLAG-tagged Trm1 was observed as a slower migrating band in lanes 3 and 4 indicated to the right of Fig 6D. Quantification using Odyssey infrared imaging revealed that the 3X-FLAG-Trm1 accumulated to about 4-fold higher than endogenous Trm1 in the same cell extracts (lanes 3, 4). We conclude that while m22G26 modification efficiency differs dramatically in EMM and YES this is not reflected by Trm1 polypeptide levels detectable by western blotting, whereas over-expression of Trm1 is readily observed.

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