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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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M22G26 hypomodification is responsible for the maf1-antisuppression paradox.A) Box plots showing misincorporations in maf1Δ, WT and maf1Δ+maf1+ strains; G26 box shows misincorporations for 36 tRNAs with G at position 26 (*paired student t test p value <0.001); the G9, A34 and A58 box plots show misincorporations for the tRNA subsets with the corresponding nucleotide identities. B) G26 misincorporations for unique reads mapping to sup-tRNASerUCA in maf1Δ, WT and maf1Δ+maf1+ strains. C) Western blot analysis of Trm1 levels in the strains indicated above the lanes; tubulin served as a loading control. D) Cartoon showing G26 as red asterisk and the two probes used for PHA26 (positive hybridization in the absence of G26 modification) assay. E) PHA26 northern blot assay showing sequential probings with oligos to the two different tRNAsLeu indicated to the left; strains are indicated above the lanes and over-expression plasmids are indicated as +trm1++maf1+ or the control, empty vector. Quantification of T-loop/D-AC stem probe signal is expressed as a modification index where the value of the control, in this case lane 1, set to a value of 1.0, is shown below the lanes of each tRNA panel. F) tRNA-mediated suppression (TMS) for WT, maf1Δ, trm1Δ, and maf1Δ+trm1+ cells.
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pgen.1005671.g004: M22G26 hypomodification is responsible for the maf1-antisuppression paradox.A) Box plots showing misincorporations in maf1Δ, WT and maf1Δ+maf1+ strains; G26 box shows misincorporations for 36 tRNAs with G at position 26 (*paired student t test p value <0.001); the G9, A34 and A58 box plots show misincorporations for the tRNA subsets with the corresponding nucleotide identities. B) G26 misincorporations for unique reads mapping to sup-tRNASerUCA in maf1Δ, WT and maf1Δ+maf1+ strains. C) Western blot analysis of Trm1 levels in the strains indicated above the lanes; tubulin served as a loading control. D) Cartoon showing G26 as red asterisk and the two probes used for PHA26 (positive hybridization in the absence of G26 modification) assay. E) PHA26 northern blot assay showing sequential probings with oligos to the two different tRNAsLeu indicated to the left; strains are indicated above the lanes and over-expression plasmids are indicated as +trm1++maf1+ or the control, empty vector. Quantification of T-loop/D-AC stem probe signal is expressed as a modification index where the value of the control, in this case lane 1, set to a value of 1.0, is shown below the lanes of each tRNA panel. F) tRNA-mediated suppression (TMS) for WT, maf1Δ, trm1Δ, and maf1Δ+trm1+ cells.

Mentions: Comparative analysis of all tRNAs at all positions in WT, maf1Δ and maf1Δ+maf1+ cells revealed that misincorporation levels at G26 were specifically altered in Trm1 target tRNAs in response to three levels of maf1+ expression in a manner that positively correlated with TMS (Fig 4A, G26 panel, compare with Fig 2A). Misincorporations detected at G9, A34 and A58, reflecting m1G9, I34 and m1A58, did not vary with maf1+ expression (Fig 4A). The specific correlation of G26 misincorporations with three levels of maf1+ expression and TMS also fits with the finding that Trm1, which appears limiting for G26 modification in WT cells (Fig 3D) may become more so as tRNA levels increase in maf1Δ cells, and as shown below, m22G26 is required for suppressor activity.


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)

M22G26 hypomodification is responsible for the maf1-antisuppression paradox.A) Box plots showing misincorporations in maf1Δ, WT and maf1Δ+maf1+ strains; G26 box shows misincorporations for 36 tRNAs with G at position 26 (*paired student t test p value <0.001); the G9, A34 and A58 box plots show misincorporations for the tRNA subsets with the corresponding nucleotide identities. B) G26 misincorporations for unique reads mapping to sup-tRNASerUCA in maf1Δ, WT and maf1Δ+maf1+ strains. C) Western blot analysis of Trm1 levels in the strains indicated above the lanes; tubulin served as a loading control. D) Cartoon showing G26 as red asterisk and the two probes used for PHA26 (positive hybridization in the absence of G26 modification) assay. E) PHA26 northern blot assay showing sequential probings with oligos to the two different tRNAsLeu indicated to the left; strains are indicated above the lanes and over-expression plasmids are indicated as +trm1++maf1+ or the control, empty vector. Quantification of T-loop/D-AC stem probe signal is expressed as a modification index where the value of the control, in this case lane 1, set to a value of 1.0, is shown below the lanes of each tRNA panel. F) tRNA-mediated suppression (TMS) for WT, maf1Δ, trm1Δ, and maf1Δ+trm1+ cells.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005671.g004: M22G26 hypomodification is responsible for the maf1-antisuppression paradox.A) Box plots showing misincorporations in maf1Δ, WT and maf1Δ+maf1+ strains; G26 box shows misincorporations for 36 tRNAs with G at position 26 (*paired student t test p value <0.001); the G9, A34 and A58 box plots show misincorporations for the tRNA subsets with the corresponding nucleotide identities. B) G26 misincorporations for unique reads mapping to sup-tRNASerUCA in maf1Δ, WT and maf1Δ+maf1+ strains. C) Western blot analysis of Trm1 levels in the strains indicated above the lanes; tubulin served as a loading control. D) Cartoon showing G26 as red asterisk and the two probes used for PHA26 (positive hybridization in the absence of G26 modification) assay. E) PHA26 northern blot assay showing sequential probings with oligos to the two different tRNAsLeu indicated to the left; strains are indicated above the lanes and over-expression plasmids are indicated as +trm1++maf1+ or the control, empty vector. Quantification of T-loop/D-AC stem probe signal is expressed as a modification index where the value of the control, in this case lane 1, set to a value of 1.0, is shown below the lanes of each tRNA panel. F) tRNA-mediated suppression (TMS) for WT, maf1Δ, trm1Δ, and maf1Δ+trm1+ cells.
Mentions: Comparative analysis of all tRNAs at all positions in WT, maf1Δ and maf1Δ+maf1+ cells revealed that misincorporation levels at G26 were specifically altered in Trm1 target tRNAs in response to three levels of maf1+ expression in a manner that positively correlated with TMS (Fig 4A, G26 panel, compare with Fig 2A). Misincorporations detected at G9, A34 and A58, reflecting m1G9, I34 and m1A58, did not vary with maf1+ expression (Fig 4A). The specific correlation of G26 misincorporations with three levels of maf1+ expression and TMS also fits with the finding that Trm1, which appears limiting for G26 modification in WT cells (Fig 3D) may become more so as tRNA levels increase in maf1Δ cells, and as shown below, m22G26 is required for suppressor activity.

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