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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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Ribosome content is reduced in yeast cells lacking IP7(A) Polysomal profiles of equal concentrations of WT and kcs1Δ yeast lysates, as measured by absorbance at 254 nm (A254). The positions of 40S and 60S ribosomal subunits, 80S monosomes and polysomes are indicated. (B) Ribosomal subunit profiles of equal concentrations of WT and kcs1Δ yeast lysates. The positions of 40S and 60S subunits are indicated. Data represent two independent experiments. (C) Total RNA isolated from yeast cells, quantified by measuring A260, was normalized to the absorbance (A600) of the culture. Data are means±S.E.M. (n=6). (D–F) Total RNA (10 μg) isolated from yeast cells was resolved using denaturing agarose gel electrophoresis. (D) 35S, 25S and 18S rRNA were quantified by densitometry analysis. (E) Levels of 35S rRNA were compared with 25S rRNA and (F) levels of 25S rRNA with 18S rRNA; these ratios in kcs1Δ cells were normalized to WT cells. Data are means±S.E.M. (n=4). P values are from (C) a two-tailed paired t-test or (E, F) a one-sample t-test (*P≤0.05; n.s. not significant, P>0.05).
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Figure 2: Ribosome content is reduced in yeast cells lacking IP7(A) Polysomal profiles of equal concentrations of WT and kcs1Δ yeast lysates, as measured by absorbance at 254 nm (A254). The positions of 40S and 60S ribosomal subunits, 80S monosomes and polysomes are indicated. (B) Ribosomal subunit profiles of equal concentrations of WT and kcs1Δ yeast lysates. The positions of 40S and 60S subunits are indicated. Data represent two independent experiments. (C) Total RNA isolated from yeast cells, quantified by measuring A260, was normalized to the absorbance (A600) of the culture. Data are means±S.E.M. (n=6). (D–F) Total RNA (10 μg) isolated from yeast cells was resolved using denaturing agarose gel electrophoresis. (D) 35S, 25S and 18S rRNA were quantified by densitometry analysis. (E) Levels of 35S rRNA were compared with 25S rRNA and (F) levels of 25S rRNA with 18S rRNA; these ratios in kcs1Δ cells were normalized to WT cells. Data are means±S.E.M. (n=4). P values are from (C) a two-tailed paired t-test or (E, F) a one-sample t-test (*P≤0.05; n.s. not significant, P>0.05).

Mentions: The lower rate of protein synthesis observed in kcs1Δ cells may be due to reduced ribosome levels or defects in ribosome activity. We therefore conducted a polysome analysis in WT and kcs1Δ yeast strains to determine the levels of monosomes and polysomes assembled on mRNA. The kcs1Δ cells display a significant reduction in monosome and polysome content (Figure 2A). Our findings are similar to those of Mizuta and colleagues, who observed that kcs1Δ yeast cells display reduced polysome levels when grown at 16°C [24], but, in contrast with their observation, we also note a decrease in 80S monosome levels in cells grown at 30°C. The decrease in polysomes could be due to a defect in the assembly of ribosomal subunits on mRNA, or reflect a decrease in total ribosome content. Under conditions in which individual ribosome subunits are detected, we noted a decrease in the levels of 40S and 60S subunits in kcs1Δ cells (Figure 2B). In a eukaryotic cell, rRNA makes up 60% of the total RNA [1,38]. Lower ribosome levels in kcs1Δ cells were therefore reflected in a 40% decrease in total RNA content (Figure 2C). However, when equal total RNA was visualized, we observed reduced levels of 35S pre-rRNA relative to mature 25S or 18S rRNA in kcs1Δ cells (Figures 2D and 2E). There was no difference in the ratio of 25S to 18S rRNA (Figure 2F), indicating that a defect in rRNA processing is unlikely to be the basis of lower ribosome levels.


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)

Ribosome content is reduced in yeast cells lacking IP7(A) Polysomal profiles of equal concentrations of WT and kcs1Δ yeast lysates, as measured by absorbance at 254 nm (A254). The positions of 40S and 60S ribosomal subunits, 80S monosomes and polysomes are indicated. (B) Ribosomal subunit profiles of equal concentrations of WT and kcs1Δ yeast lysates. The positions of 40S and 60S subunits are indicated. Data represent two independent experiments. (C) Total RNA isolated from yeast cells, quantified by measuring A260, was normalized to the absorbance (A600) of the culture. Data are means±S.E.M. (n=6). (D–F) Total RNA (10 μg) isolated from yeast cells was resolved using denaturing agarose gel electrophoresis. (D) 35S, 25S and 18S rRNA were quantified by densitometry analysis. (E) Levels of 35S rRNA were compared with 25S rRNA and (F) levels of 25S rRNA with 18S rRNA; these ratios in kcs1Δ cells were normalized to WT cells. Data are means±S.E.M. (n=4). P values are from (C) a two-tailed paired t-test or (E, F) a one-sample t-test (*P≤0.05; n.s. not significant, P>0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Ribosome content is reduced in yeast cells lacking IP7(A) Polysomal profiles of equal concentrations of WT and kcs1Δ yeast lysates, as measured by absorbance at 254 nm (A254). The positions of 40S and 60S ribosomal subunits, 80S monosomes and polysomes are indicated. (B) Ribosomal subunit profiles of equal concentrations of WT and kcs1Δ yeast lysates. The positions of 40S and 60S subunits are indicated. Data represent two independent experiments. (C) Total RNA isolated from yeast cells, quantified by measuring A260, was normalized to the absorbance (A600) of the culture. Data are means±S.E.M. (n=6). (D–F) Total RNA (10 μg) isolated from yeast cells was resolved using denaturing agarose gel electrophoresis. (D) 35S, 25S and 18S rRNA were quantified by densitometry analysis. (E) Levels of 35S rRNA were compared with 25S rRNA and (F) levels of 25S rRNA with 18S rRNA; these ratios in kcs1Δ cells were normalized to WT cells. Data are means±S.E.M. (n=4). P values are from (C) a two-tailed paired t-test or (E, F) a one-sample t-test (*P≤0.05; n.s. not significant, P>0.05).
Mentions: The lower rate of protein synthesis observed in kcs1Δ cells may be due to reduced ribosome levels or defects in ribosome activity. We therefore conducted a polysome analysis in WT and kcs1Δ yeast strains to determine the levels of monosomes and polysomes assembled on mRNA. The kcs1Δ cells display a significant reduction in monosome and polysome content (Figure 2A). Our findings are similar to those of Mizuta and colleagues, who observed that kcs1Δ yeast cells display reduced polysome levels when grown at 16°C [24], but, in contrast with their observation, we also note a decrease in 80S monosome levels in cells grown at 30°C. The decrease in polysomes could be due to a defect in the assembly of ribosomal subunits on mRNA, or reflect a decrease in total ribosome content. Under conditions in which individual ribosome subunits are detected, we noted a decrease in the levels of 40S and 60S subunits in kcs1Δ cells (Figure 2B). In a eukaryotic cell, rRNA makes up 60% of the total RNA [1,38]. Lower ribosome levels in kcs1Δ cells were therefore reflected in a 40% decrease in total RNA content (Figure 2C). However, when equal total RNA was visualized, we observed reduced levels of 35S pre-rRNA relative to mature 25S or 18S rRNA in kcs1Δ cells (Figures 2D and 2E). There was no difference in the ratio of 25S to 18S rRNA (Figure 2F), indicating that a defect in rRNA processing is unlikely to be the basis of lower ribosome levels.

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