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Protein degradation and dynamic tRNA thiolation fine-tune translation at elevated temperatures.

Tyagi K, Pedrioli PG - Nucleic Acids Res. (2015)

Bottom Line: In agreement with previous transcriptomics studies, amongst the most marked changes, we found up-regulation of cytoprotective factors; a shift from oxidative phosphorylation to fermentation; and down-regulation of translation.Using random forests we show that this results in differential translation of transcripts with a biased content for the corresponding codons.We propose that the role of this pathway in promoting catabolic and inhibiting anabolic processes, affords cells with additional time and resources needed to attain proper protein folding under periods of stress.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

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Independent mechanisms regulate tRNA thiolation upon S-AA starvation and growth at elevated temperature. (A) Relative changes in abundance of nucleosides as measured by RNA-MS analysis. tRNAs were either purified from cells starved for S-AA (no S-AA) or in the presence of methionine (+Met). Adenosine is shown as a control. (B) APM-dPAGE or northern blot analyses indicate a similar reduction in thiolation in tRNA isolated from cells starved for S-AA or grown at elevated temperature. (C) npr2Δ prevents hypothiolation in cells starved for S-AA, but not in cells grown at 37°C. (D) Hypothiolation at 37°C does not depend on S-AA content. (E) ncs2_A212T prevents hypothiolation at 37°C, but not in S-AA starved cells. (F) Mean ΔCt values for three biological replicates of quantitative real time PCR measurements for URM1-pathway mRNA levels in cells grown at 37°C or 30°C. The mean ΔCt value of IPP1 was used for normalization. 18S rRNA and ACT1 were analysed as negative controls. (*) and (**) indicate P-values of less than 0.05 and 0.01, respectively, obtained by a two-sample t-test of the difference between the ΔCt values at 30°C versus 37°C for a given gene. Error bars represent standard error (n = 3). (G) APM-dPAGE analysis for tRNA extracted from wild-type, cim5-1 and cim3-1 yeast cells grown at either 30°C or 37°C.
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Figure 4: Independent mechanisms regulate tRNA thiolation upon S-AA starvation and growth at elevated temperature. (A) Relative changes in abundance of nucleosides as measured by RNA-MS analysis. tRNAs were either purified from cells starved for S-AA (no S-AA) or in the presence of methionine (+Met). Adenosine is shown as a control. (B) APM-dPAGE or northern blot analyses indicate a similar reduction in thiolation in tRNA isolated from cells starved for S-AA or grown at elevated temperature. (C) npr2Δ prevents hypothiolation in cells starved for S-AA, but not in cells grown at 37°C. (D) Hypothiolation at 37°C does not depend on S-AA content. (E) ncs2_A212T prevents hypothiolation at 37°C, but not in S-AA starved cells. (F) Mean ΔCt values for three biological replicates of quantitative real time PCR measurements for URM1-pathway mRNA levels in cells grown at 37°C or 30°C. The mean ΔCt value of IPP1 was used for normalization. 18S rRNA and ACT1 were analysed as negative controls. (*) and (**) indicate P-values of less than 0.05 and 0.01, respectively, obtained by a two-sample t-test of the difference between the ΔCt values at 30°C versus 37°C for a given gene. Error bars represent standard error (n = 3). (G) APM-dPAGE analysis for tRNA extracted from wild-type, cim5-1 and cim3-1 yeast cells grown at either 30°C or 37°C.

Mentions: Laxman et al. (45) have recently shown that tRNA hypothiolation in cells starved for sulfur containing amino acids (S-AA) can be rescued by deleting NPR2. To test if hypothiolation in cells grown at elevated temperatures is controlled in a similar fashion, we first analysed the down-regulation of thiolation in S-AA starved cells by RNA-MS and northern blot analysis (Figure 4A and B). This indicated that S-AA starvation and growth at elevated temperatures had similar effects on tRNA modification. However, although S-AA starvation induced tRNA hypothiolation was effectively suppressed in an npr2Δ strain (Figure 4C compare change in thiolation level between lanes 1, 2 (npr2Δ background) to that between lanes 4, 5 (wt background)), the same was not the case for temperature induced hypothiolation (Figure 4C compare change in thiolation level between lanes 1, 3 (npr2Δ background) to that between lanes 4, 6 (wt background)). Furthermore, the latter was not rescued by methionine supplementation, nor by growing the cells in a complex rich media (Figure 4D).


Protein degradation and dynamic tRNA thiolation fine-tune translation at elevated temperatures.

Tyagi K, Pedrioli PG - Nucleic Acids Res. (2015)

Independent mechanisms regulate tRNA thiolation upon S-AA starvation and growth at elevated temperature. (A) Relative changes in abundance of nucleosides as measured by RNA-MS analysis. tRNAs were either purified from cells starved for S-AA (no S-AA) or in the presence of methionine (+Met). Adenosine is shown as a control. (B) APM-dPAGE or northern blot analyses indicate a similar reduction in thiolation in tRNA isolated from cells starved for S-AA or grown at elevated temperature. (C) npr2Δ prevents hypothiolation in cells starved for S-AA, but not in cells grown at 37°C. (D) Hypothiolation at 37°C does not depend on S-AA content. (E) ncs2_A212T prevents hypothiolation at 37°C, but not in S-AA starved cells. (F) Mean ΔCt values for three biological replicates of quantitative real time PCR measurements for URM1-pathway mRNA levels in cells grown at 37°C or 30°C. The mean ΔCt value of IPP1 was used for normalization. 18S rRNA and ACT1 were analysed as negative controls. (*) and (**) indicate P-values of less than 0.05 and 0.01, respectively, obtained by a two-sample t-test of the difference between the ΔCt values at 30°C versus 37°C for a given gene. Error bars represent standard error (n = 3). (G) APM-dPAGE analysis for tRNA extracted from wild-type, cim5-1 and cim3-1 yeast cells grown at either 30°C or 37°C.
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Figure 4: Independent mechanisms regulate tRNA thiolation upon S-AA starvation and growth at elevated temperature. (A) Relative changes in abundance of nucleosides as measured by RNA-MS analysis. tRNAs were either purified from cells starved for S-AA (no S-AA) or in the presence of methionine (+Met). Adenosine is shown as a control. (B) APM-dPAGE or northern blot analyses indicate a similar reduction in thiolation in tRNA isolated from cells starved for S-AA or grown at elevated temperature. (C) npr2Δ prevents hypothiolation in cells starved for S-AA, but not in cells grown at 37°C. (D) Hypothiolation at 37°C does not depend on S-AA content. (E) ncs2_A212T prevents hypothiolation at 37°C, but not in S-AA starved cells. (F) Mean ΔCt values for three biological replicates of quantitative real time PCR measurements for URM1-pathway mRNA levels in cells grown at 37°C or 30°C. The mean ΔCt value of IPP1 was used for normalization. 18S rRNA and ACT1 were analysed as negative controls. (*) and (**) indicate P-values of less than 0.05 and 0.01, respectively, obtained by a two-sample t-test of the difference between the ΔCt values at 30°C versus 37°C for a given gene. Error bars represent standard error (n = 3). (G) APM-dPAGE analysis for tRNA extracted from wild-type, cim5-1 and cim3-1 yeast cells grown at either 30°C or 37°C.
Mentions: Laxman et al. (45) have recently shown that tRNA hypothiolation in cells starved for sulfur containing amino acids (S-AA) can be rescued by deleting NPR2. To test if hypothiolation in cells grown at elevated temperatures is controlled in a similar fashion, we first analysed the down-regulation of thiolation in S-AA starved cells by RNA-MS and northern blot analysis (Figure 4A and B). This indicated that S-AA starvation and growth at elevated temperatures had similar effects on tRNA modification. However, although S-AA starvation induced tRNA hypothiolation was effectively suppressed in an npr2Δ strain (Figure 4C compare change in thiolation level between lanes 1, 2 (npr2Δ background) to that between lanes 4, 5 (wt background)), the same was not the case for temperature induced hypothiolation (Figure 4C compare change in thiolation level between lanes 1, 3 (npr2Δ background) to that between lanes 4, 6 (wt background)). Furthermore, the latter was not rescued by methionine supplementation, nor by growing the cells in a complex rich media (Figure 4D).

Bottom Line: In agreement with previous transcriptomics studies, amongst the most marked changes, we found up-regulation of cytoprotective factors; a shift from oxidative phosphorylation to fermentation; and down-regulation of translation.Using random forests we show that this results in differential translation of transcripts with a biased content for the corresponding codons.We propose that the role of this pathway in promoting catabolic and inhibiting anabolic processes, affords cells with additional time and resources needed to attain proper protein folding under periods of stress.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

Show MeSH
Related in: MedlinePlus