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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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S. cerevisiae lacking Kcs1 displays a defect in translation(A) 5-fold serial dilutions of the indicated S. cerevisiae strains were plated on YPD with or without protein synthesis inhibitors, and incubated for 2–3 days at 30°C. Data represent three independent experiments. (B) Protein synthesis was measured in the indicated S. cerevisiae strains by pulse-labelling cells for 5 min with [35S]methionine/cysteine. Radioactivity incorporated into total protein, expressed as counts per min (cpm), was normalized to the absorbance (A600) of the labelled culture. Data are means±S.E.M. (n=4). (C) Protein synthesis was measured in kcs1Δ cells expressing either native or catalytically inactive forms of Kcs1, as described in (B). Data are means±S.E.M. (n=6). P values are from a two-tailed paired t-test (*P≤0.05; **P≤0.01; n.s. not significant, P>0.05).
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Figure 1: S. cerevisiae lacking Kcs1 displays a defect in translation(A) 5-fold serial dilutions of the indicated S. cerevisiae strains were plated on YPD with or without protein synthesis inhibitors, and incubated for 2–3 days at 30°C. Data represent three independent experiments. (B) Protein synthesis was measured in the indicated S. cerevisiae strains by pulse-labelling cells for 5 min with [35S]methionine/cysteine. Radioactivity incorporated into total protein, expressed as counts per min (cpm), was normalized to the absorbance (A600) of the labelled culture. Data are means±S.E.M. (n=4). (C) Protein synthesis was measured in kcs1Δ cells expressing either native or catalytically inactive forms of Kcs1, as described in (B). Data are means±S.E.M. (n=6). P values are from a two-tailed paired t-test (*P≤0.05; **P≤0.01; n.s. not significant, P>0.05).

Mentions: We monitored ribosome function in yeast cells by growing them in the presence of aminoglycoside antibiotics, G418, paromomycin or hygromycin B, which disrupt the elongation cycle during polypeptide synthesis. We observed that kcs1Δ yeast cells, which have no detectable inositol pyrophosphates, are sensitive to low doses of these drugs; at these doses WT yeast cells show either no change in growth or mild sensitivity (Figure 1A). Conversely, vip1Δ yeast cells, which have higher levels of 5-IP7 than WT yeast cells but no 1-IP7, show no loss of viability at low doses of these inhibitors. We also tested the double knockout kcs1Δddp1Δ strain, which accumulates 1-IP7 but has no 5-IP7 or IP8. The additional removal of Ddp1 partially rescues the sensitivity of yeast cells lacking Kcs1 to G418, and completely eliminates sensitivity to paromomycin and hygromycin B. This suggests that the presence of either 1-IP7 or 5-IP7 is sufficient to confer resistance to translation inhibitors. The sensitivity of kcs1Δ yeast strain to translation inhibitors could reflect a defect in protein synthesis. Incorporation of radiolabelled methionine/cysteine into proteins is significantly reduced in kcs1Δ yeast cells compared with WT cells (Figure 1B), but, as expected, vip1Δ yeast cells show no change in the rate of protein synthesis.


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)

S. cerevisiae lacking Kcs1 displays a defect in translation(A) 5-fold serial dilutions of the indicated S. cerevisiae strains were plated on YPD with or without protein synthesis inhibitors, and incubated for 2–3 days at 30°C. Data represent three independent experiments. (B) Protein synthesis was measured in the indicated S. cerevisiae strains by pulse-labelling cells for 5 min with [35S]methionine/cysteine. Radioactivity incorporated into total protein, expressed as counts per min (cpm), was normalized to the absorbance (A600) of the labelled culture. Data are means±S.E.M. (n=4). (C) Protein synthesis was measured in kcs1Δ cells expressing either native or catalytically inactive forms of Kcs1, as described in (B). Data are means±S.E.M. (n=6). P values are from a two-tailed paired t-test (*P≤0.05; **P≤0.01; n.s. not significant, P>0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: S. cerevisiae lacking Kcs1 displays a defect in translation(A) 5-fold serial dilutions of the indicated S. cerevisiae strains were plated on YPD with or without protein synthesis inhibitors, and incubated for 2–3 days at 30°C. Data represent three independent experiments. (B) Protein synthesis was measured in the indicated S. cerevisiae strains by pulse-labelling cells for 5 min with [35S]methionine/cysteine. Radioactivity incorporated into total protein, expressed as counts per min (cpm), was normalized to the absorbance (A600) of the labelled culture. Data are means±S.E.M. (n=4). (C) Protein synthesis was measured in kcs1Δ cells expressing either native or catalytically inactive forms of Kcs1, as described in (B). Data are means±S.E.M. (n=6). P values are from a two-tailed paired t-test (*P≤0.05; **P≤0.01; n.s. not significant, P>0.05).
Mentions: We monitored ribosome function in yeast cells by growing them in the presence of aminoglycoside antibiotics, G418, paromomycin or hygromycin B, which disrupt the elongation cycle during polypeptide synthesis. We observed that kcs1Δ yeast cells, which have no detectable inositol pyrophosphates, are sensitive to low doses of these drugs; at these doses WT yeast cells show either no change in growth or mild sensitivity (Figure 1A). Conversely, vip1Δ yeast cells, which have higher levels of 5-IP7 than WT yeast cells but no 1-IP7, show no loss of viability at low doses of these inhibitors. We also tested the double knockout kcs1Δddp1Δ strain, which accumulates 1-IP7 but has no 5-IP7 or IP8. The additional removal of Ddp1 partially rescues the sensitivity of yeast cells lacking Kcs1 to G418, and completely eliminates sensitivity to paromomycin and hygromycin B. This suggests that the presence of either 1-IP7 or 5-IP7 is sufficient to confer resistance to translation inhibitors. The sensitivity of kcs1Δ yeast strain to translation inhibitors could reflect a defect in protein synthesis. Incorporation of radiolabelled methionine/cysteine into proteins is significantly reduced in kcs1Δ yeast cells compared with WT cells (Figure 1B), but, as expected, vip1Δ yeast cells show no change in the rate of protein synthesis.

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