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Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae.

Thota SG, Unnikannan CP, Thampatty SR, Manorama R, Bhandari R - Biochem. J. (2015)

Bottom Line: We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae.We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation.Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

View Article: PubMed Central - PubMed

Affiliation: *Laboratory of Cell Signalling, Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad, Telangana, India.

ABSTRACT
Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1Δ yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1Δ yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1Δ yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

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Synthesis of rRNA is reduced in yeast cells lacking IP7(A) Yeast cells were labelled with [14C]uracil for the time indicated; RNA was isolated and resolved using denaturing agarose gel electrophoresis, detected by staining with ethidium bromide (lower panel) and transferred to a nylon membrane. Incorporation of radiolabelled uracil into rRNA was detected by phosphorimager scanning (upper panel). Data represent two independent experiments. (B) For the experiment described in (A), [14C]uracil incorporation into 18S rRNA (intensity of phosphorimager signal) was normalized to total 18S rRNA (intensity of ethidium bromide staining) at 5, 15 and 20 min, and these ratios in kcs1Δ cells were compared with those in WT cells. Data are means±S.E.M. (n=6). P values are from a one-sample t-test (***P≤0.001). (C) Yeast cells were labelled with [14C]uracil for 5 min and chased with excess unlabelled uracil for the time indicated. RNA was isolated, equal amounts of total RNA were resolved using denaturing agarose gel electrophoresis and the radioactivity was detected with phosphorimager scanning. The kcs1Δ blot, which had a fainter signal compared with the WT blot, was subjected to linear contrast adjustment to visualize bands. Data represent two independent experiments.
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Figure 3: Synthesis of rRNA is reduced in yeast cells lacking IP7(A) Yeast cells were labelled with [14C]uracil for the time indicated; RNA was isolated and resolved using denaturing agarose gel electrophoresis, detected by staining with ethidium bromide (lower panel) and transferred to a nylon membrane. Incorporation of radiolabelled uracil into rRNA was detected by phosphorimager scanning (upper panel). Data represent two independent experiments. (B) For the experiment described in (A), [14C]uracil incorporation into 18S rRNA (intensity of phosphorimager signal) was normalized to total 18S rRNA (intensity of ethidium bromide staining) at 5, 15 and 20 min, and these ratios in kcs1Δ cells were compared with those in WT cells. Data are means±S.E.M. (n=6). P values are from a one-sample t-test (***P≤0.001). (C) Yeast cells were labelled with [14C]uracil for 5 min and chased with excess unlabelled uracil for the time indicated. RNA was isolated, equal amounts of total RNA were resolved using denaturing agarose gel electrophoresis and the radioactivity was detected with phosphorimager scanning. The kcs1Δ blot, which had a fainter signal compared with the WT blot, was subjected to linear contrast adjustment to visualize bands. Data represent two independent experiments.

Mentions: Reduced 35S rRNA levels may result from a reduction in rRNA synthesis by RNA Pol I. We examined rRNA synthesis by labelling cells for different lengths of time with [14C]uracil. The kcs1Δ cells display a substantial reduction in the incorporation of radiolabelled uracil into rRNA when normalized for total rRNA (Figures 3A and 3B). To track pre-rRNA processing, we pulse labelled cells with [14C]uracil for 5 min, and monitored radiolabelled rRNA levels during a chase with unlabelled uracil for different lengths of time. There was no accumulation of 27S and 20S precursor rRNAs in kcs1Δ cells, and no apparent difference in the rates of pre-rRNA processing to form mature 25S and 18S rRNA (Figure 3C). These data suggest that a reduction in rRNA synthesis may be the basis for the sensitivity to aminoglycoside antibiotics, lower ribosome level and reduced growth rate observed in yeasts lacking inositol pyrophosphates.


Inositol pyrophosphates regulate RNA polymerase I-mediated rRNA transcription in Saccharomyces cerevisiae.

Thota SG, Unnikannan CP, Thampatty SR, Manorama R, Bhandari R - Biochem. J. (2015)

Synthesis of rRNA is reduced in yeast cells lacking IP7(A) Yeast cells were labelled with [14C]uracil for the time indicated; RNA was isolated and resolved using denaturing agarose gel electrophoresis, detected by staining with ethidium bromide (lower panel) and transferred to a nylon membrane. Incorporation of radiolabelled uracil into rRNA was detected by phosphorimager scanning (upper panel). Data represent two independent experiments. (B) For the experiment described in (A), [14C]uracil incorporation into 18S rRNA (intensity of phosphorimager signal) was normalized to total 18S rRNA (intensity of ethidium bromide staining) at 5, 15 and 20 min, and these ratios in kcs1Δ cells were compared with those in WT cells. Data are means±S.E.M. (n=6). P values are from a one-sample t-test (***P≤0.001). (C) Yeast cells were labelled with [14C]uracil for 5 min and chased with excess unlabelled uracil for the time indicated. RNA was isolated, equal amounts of total RNA were resolved using denaturing agarose gel electrophoresis and the radioactivity was detected with phosphorimager scanning. The kcs1Δ blot, which had a fainter signal compared with the WT blot, was subjected to linear contrast adjustment to visualize bands. Data represent two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4325516&req=5

Figure 3: Synthesis of rRNA is reduced in yeast cells lacking IP7(A) Yeast cells were labelled with [14C]uracil for the time indicated; RNA was isolated and resolved using denaturing agarose gel electrophoresis, detected by staining with ethidium bromide (lower panel) and transferred to a nylon membrane. Incorporation of radiolabelled uracil into rRNA was detected by phosphorimager scanning (upper panel). Data represent two independent experiments. (B) For the experiment described in (A), [14C]uracil incorporation into 18S rRNA (intensity of phosphorimager signal) was normalized to total 18S rRNA (intensity of ethidium bromide staining) at 5, 15 and 20 min, and these ratios in kcs1Δ cells were compared with those in WT cells. Data are means±S.E.M. (n=6). P values are from a one-sample t-test (***P≤0.001). (C) Yeast cells were labelled with [14C]uracil for 5 min and chased with excess unlabelled uracil for the time indicated. RNA was isolated, equal amounts of total RNA were resolved using denaturing agarose gel electrophoresis and the radioactivity was detected with phosphorimager scanning. The kcs1Δ blot, which had a fainter signal compared with the WT blot, was subjected to linear contrast adjustment to visualize bands. Data represent two independent experiments.
Mentions: Reduced 35S rRNA levels may result from a reduction in rRNA synthesis by RNA Pol I. We examined rRNA synthesis by labelling cells for different lengths of time with [14C]uracil. The kcs1Δ cells display a substantial reduction in the incorporation of radiolabelled uracil into rRNA when normalized for total rRNA (Figures 3A and 3B). To track pre-rRNA processing, we pulse labelled cells with [14C]uracil for 5 min, and monitored radiolabelled rRNA levels during a chase with unlabelled uracil for different lengths of time. There was no accumulation of 27S and 20S precursor rRNAs in kcs1Δ cells, and no apparent difference in the rates of pre-rRNA processing to form mature 25S and 18S rRNA (Figure 3C). These data suggest that a reduction in rRNA synthesis may be the basis for the sensitivity to aminoglycoside antibiotics, lower ribosome level and reduced growth rate observed in yeasts lacking inositol pyrophosphates.

Bottom Line: We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae.We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation.Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

View Article: PubMed Central - PubMed

Affiliation: *Laboratory of Cell Signalling, Centre for DNA Fingerprinting and Diagnostics, Nampally, Hyderabad, Telangana, India.

ABSTRACT
Ribosome biogenesis is an essential cellular process regulated by the metabolic state of a cell. We examined whether inositol pyrophosphates, energy-rich derivatives of inositol that act as metabolic messengers, play a role in ribosome synthesis in the budding yeast, Saccharomyces cerevisiae. Yeast strains lacking the inositol hexakisphosphate (IP6) kinase Kcs1, which is required for the synthesis of inositol pyrophosphates, display increased sensitivity to translation inhibitors and decreased protein synthesis. These phenotypes are reversed on expression of enzymatically active Kcs1, but not on expression of the inactive form. The kcs1Δ yeast cells exhibit reduced levels of ribosome subunits, suggesting that they are defective in ribosome biogenesis. The rate of rRNA synthesis, the first step of ribosome biogenesis, is decreased in kcs1Δ yeast strains, suggesting that RNA polymerase I (Pol I) activity may be reduced in these cells. We determined that the Pol I subunits, A190, A43 and A34.5, can accept a β-phosphate moiety from inositol pyrophosphates to undergo serine pyrophosphorylation. Although there is impaired rRNA synthesis in kcs1Δ yeast cells, we did not find any defect in recruitment of Pol I on rDNA, but observed that the rate of transcription elongation was compromised. Taken together, our findings highlight inositol pyrophosphates as novel regulators of rRNA transcription.

Show MeSH
Related in: MedlinePlus