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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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RNA Pol I elongation activity is lowered in kcs1Δ yeast strain(A) The 9.1-kb transcription unit of rDNA includes a 6.6-kb region encoding 35S pre-rRNA transcribed by RNA Pol I, a 121-bp region encoding 5S rRNA transcribed by RNA Pol III from the opposite strand and two NTSs. The 35S rDNA consists of 5′- and 3′-ETSs, two ITSs), and regions encoding the 18S, 5.8S and 25S mature rRNAs. Primers used for quantitative PCR (qPCR) are indicated by arrows. Primers 1 and 2 amplify the rDNA promoter (−174 to +57), and primers 3 and 4 amplify the 5′-ETS (+91 to +270). Probes used for transcription run-on analysis are indicated by solid lines. (B) Chromatin immunoprecipitation with GST-tagged A43, followed by qPCR with primers indicated in (A). Immunoprecipitated chromatin is expressed as a percentage of input chromatin in each sample. Data are means±S.E.M. (n=3). (C) Transcription run-on analysis using probes indicated in (A). The hybridization signals were quantified by densitometry analysis; the average intensity of the TOPO spots was considered as a background, and individual probe intensities were normalized to the genomic DNA signal. (D) These ratios in kcs1Δ cells were normalized to WT cells. Data are means±S.E.M. (n=4). P values are from (B) a two-tailed paired t-test or (D) a one-sample t-test (*P≤0.05; **P≤0.01; n.s. not significant, P>0.05).
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Figure 5: RNA Pol I elongation activity is lowered in kcs1Δ yeast strain(A) The 9.1-kb transcription unit of rDNA includes a 6.6-kb region encoding 35S pre-rRNA transcribed by RNA Pol I, a 121-bp region encoding 5S rRNA transcribed by RNA Pol III from the opposite strand and two NTSs. The 35S rDNA consists of 5′- and 3′-ETSs, two ITSs), and regions encoding the 18S, 5.8S and 25S mature rRNAs. Primers used for quantitative PCR (qPCR) are indicated by arrows. Primers 1 and 2 amplify the rDNA promoter (−174 to +57), and primers 3 and 4 amplify the 5′-ETS (+91 to +270). Probes used for transcription run-on analysis are indicated by solid lines. (B) Chromatin immunoprecipitation with GST-tagged A43, followed by qPCR with primers indicated in (A). Immunoprecipitated chromatin is expressed as a percentage of input chromatin in each sample. Data are means±S.E.M. (n=3). (C) Transcription run-on analysis using probes indicated in (A). The hybridization signals were quantified by densitometry analysis; the average intensity of the TOPO spots was considered as a background, and individual probe intensities were normalized to the genomic DNA signal. (D) These ratios in kcs1Δ cells were normalized to WT cells. Data are means±S.E.M. (n=4). P values are from (B) a two-tailed paired t-test or (D) a one-sample t-test (*P≤0.05; **P≤0.01; n.s. not significant, P>0.05).

Mentions: The absence of IP7 may affect Pol I binding to the rDNA promoter, transcription initiation or elongation. S. cerevisiae has approximately 150 copies of tandem rDNA units arranged on chromosome XII, of which approximately half are transcriptionally active in exponentially growing cells [2]. Each rDNA unit encodes a 35S pre-rRNA transcribed by Pol I, which can be divided into regions coding for the mature 18S, 5.8S and 25S rRNA, two ETSs and two internal transcribed spacers (ITSs) which are cleaved during 35S pre-rRNA processing (Figure 5A). We used chromatin immunoprecipitation assays to monitor recruitment of the Pol I complex to the rDNA promoter. There is no difference in promoter binding by Pol I in WT and kcs1Δ cells (Figure 5B). To examine the levels of active elongating Pol I, we measured Pol I bound to the 5′-ETS, which occurs approximately 200 bp downstream of the promoter. There is no significant difference in Pol I occupancy of this region of the rDNA locus in WT and kcs1Δ yeast strains (Figure 5B). These results suggest that IP7 does not influence the recruitment of Pol I to the rDNA locus.


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)

RNA Pol I elongation activity is lowered in kcs1Δ yeast strain(A) The 9.1-kb transcription unit of rDNA includes a 6.6-kb region encoding 35S pre-rRNA transcribed by RNA Pol I, a 121-bp region encoding 5S rRNA transcribed by RNA Pol III from the opposite strand and two NTSs. The 35S rDNA consists of 5′- and 3′-ETSs, two ITSs), and regions encoding the 18S, 5.8S and 25S mature rRNAs. Primers used for quantitative PCR (qPCR) are indicated by arrows. Primers 1 and 2 amplify the rDNA promoter (−174 to +57), and primers 3 and 4 amplify the 5′-ETS (+91 to +270). Probes used for transcription run-on analysis are indicated by solid lines. (B) Chromatin immunoprecipitation with GST-tagged A43, followed by qPCR with primers indicated in (A). Immunoprecipitated chromatin is expressed as a percentage of input chromatin in each sample. Data are means±S.E.M. (n=3). (C) Transcription run-on analysis using probes indicated in (A). The hybridization signals were quantified by densitometry analysis; the average intensity of the TOPO spots was considered as a background, and individual probe intensities were normalized to the genomic DNA signal. (D) These ratios in kcs1Δ cells were normalized to WT cells. Data are means±S.E.M. (n=4). P values are from (B) a two-tailed paired t-test or (D) a one-sample t-test (*P≤0.05; **P≤0.01; n.s. not significant, P>0.05).
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Figure 5: RNA Pol I elongation activity is lowered in kcs1Δ yeast strain(A) The 9.1-kb transcription unit of rDNA includes a 6.6-kb region encoding 35S pre-rRNA transcribed by RNA Pol I, a 121-bp region encoding 5S rRNA transcribed by RNA Pol III from the opposite strand and two NTSs. The 35S rDNA consists of 5′- and 3′-ETSs, two ITSs), and regions encoding the 18S, 5.8S and 25S mature rRNAs. Primers used for quantitative PCR (qPCR) are indicated by arrows. Primers 1 and 2 amplify the rDNA promoter (−174 to +57), and primers 3 and 4 amplify the 5′-ETS (+91 to +270). Probes used for transcription run-on analysis are indicated by solid lines. (B) Chromatin immunoprecipitation with GST-tagged A43, followed by qPCR with primers indicated in (A). Immunoprecipitated chromatin is expressed as a percentage of input chromatin in each sample. Data are means±S.E.M. (n=3). (C) Transcription run-on analysis using probes indicated in (A). The hybridization signals were quantified by densitometry analysis; the average intensity of the TOPO spots was considered as a background, and individual probe intensities were normalized to the genomic DNA signal. (D) These ratios in kcs1Δ cells were normalized to WT cells. Data are means±S.E.M. (n=4). P values are from (B) a two-tailed paired t-test or (D) a one-sample t-test (*P≤0.05; **P≤0.01; n.s. not significant, P>0.05).
Mentions: The absence of IP7 may affect Pol I binding to the rDNA promoter, transcription initiation or elongation. S. cerevisiae has approximately 150 copies of tandem rDNA units arranged on chromosome XII, of which approximately half are transcriptionally active in exponentially growing cells [2]. Each rDNA unit encodes a 35S pre-rRNA transcribed by Pol I, which can be divided into regions coding for the mature 18S, 5.8S and 25S rRNA, two ETSs and two internal transcribed spacers (ITSs) which are cleaved during 35S pre-rRNA processing (Figure 5A). We used chromatin immunoprecipitation assays to monitor recruitment of the Pol I complex to the rDNA promoter. There is no difference in promoter binding by Pol I in WT and kcs1Δ cells (Figure 5B). To examine the levels of active elongating Pol I, we measured Pol I bound to the 5′-ETS, which occurs approximately 200 bp downstream of the promoter. There is no significant difference in Pol I occupancy of this region of the rDNA locus in WT and kcs1Δ yeast strains (Figure 5B). These results suggest that IP7 does not influence the recruitment of Pol I to the rDNA locus.

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