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Ccr4-not regulates RNA polymerase I transcription and couples nutrient signaling to the control of ribosomal RNA biogenesis.

Laribee RN, Hosni-Ahmed A, Workman JJ, Chen H - PLoS Genet. (2015)

Bottom Line: Furthermore, ccr4Δ is synthetically sick when paired with rpa12Δ and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation.Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation.Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis.

View Article: PubMed Central - PubMed

Affiliation: University of Tennessee Health Science Center Department of Pathology and Laboratory Medicine and the UT Center for Cancer Research, Memphis, Tennessee, United States of America.

ABSTRACT
Ribosomal RNA synthesis is controlled by nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 regulates ribosomal RNA expression by affecting RNA Polymerase I (Pol I)-dependent transcription of the ribosomal DNA (rDNA) but the mechanisms involved remain obscure. This study provides evidence that the Ccr4-Not complex, which regulates RNA Polymerase II (Pol II) transcription, also functions downstream of mTORC1 to control Pol I activity. Ccr4-Not localizes to the rDNA and physically associates with the Pol I holoenzyme while Ccr4-Not disruption perturbs rDNA binding of multiple Pol I transcriptional regulators including core factor, the high mobility group protein Hmo1, and the SSU processome. Under nutrient rich conditions, Ccr4-Not suppresses Pol I initiation by regulating interactions with the essential transcription factor Rrn3. Additionally, Ccr4-Not disruption prevents reduced Pol I transcription when mTORC1 is inhibited suggesting Ccr4-Not bridges mTORC1 signaling with Pol I regulation. Analysis of the non-essential Pol I subunits demonstrated that the A34.5 subunit promotes, while the A12.2 and A14 subunits repress, Ccr4-Not interactions with Pol I. Furthermore, ccr4Δ is synthetically sick when paired with rpa12Δ and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation. Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation. Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis.

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Ccr4-Not binds the rDNA and interacts with RNA Pol I.(A) Schematic of the rDNA locus. The relative position of the qPCR primers used are shown below as black bars. (B) ChIP of Ccr4-TAP, Not1-TAP, and Not2-TAP at the rDNA. The results are the average and standard deviation (SD) of three to four independent experiments. (C) Ccr4 associates with Pol I. 500 μg of cell extract from the indicated strains was utilized for α-HA immunoprecipitation and the presence of Ccr4 determined by α-Myc immunoblot. Statistical significance was determined by Student’s t-test. *- p< 0.05; **- p<0.01.
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pgen.1005113.g001: Ccr4-Not binds the rDNA and interacts with RNA Pol I.(A) Schematic of the rDNA locus. The relative position of the qPCR primers used are shown below as black bars. (B) ChIP of Ccr4-TAP, Not1-TAP, and Not2-TAP at the rDNA. The results are the average and standard deviation (SD) of three to four independent experiments. (C) Ccr4 associates with Pol I. 500 μg of cell extract from the indicated strains was utilized for α-HA immunoprecipitation and the presence of Ccr4 determined by α-Myc immunoblot. Statistical significance was determined by Student’s t-test. *- p< 0.05; **- p<0.01.

Mentions: Specific Ccr4-Not mutants result in significant growth defects which prompted us to ask whether Ccr4-Not might contribute to Pol I transcription since rRNA biogenesis is rate limiting for cell growth and proliferation [35]. Utilizing yeast strains carrying C-terminal TAP epitope tags integrated at either the CCR4, NOT1, or NOT2 genomic loci, we cultured cells in nutrient rich media (YPD) and performed chromatin immunoprecipitation (ChIP) coupled with quantitative PCR utilizing primers to the rDNA locus as outlined (Fig. 1A). All three Ccr4-Not subunits were significantly enriched above background suggesting that Ccr4-Not physically localizes to rDNA repeats (Fig. 1B) [43]. Next, we engineered strains to express either Rpa190-6XHA (Pol I catalytic subunit), Ccr4-13XMyc, or both, and performed co-immunoprecipitation (co-IP) experiments to determine if Ccr4-Not associates with Pol I. Precipitation of Pol I readily co-precipitated Ccr4, suggesting these complexes physically interact (Fig. 1C). Collectively, these data are consistent with the possibility that Ccr4-Not directly regulates Pol I transcription.


Ccr4-not regulates RNA polymerase I transcription and couples nutrient signaling to the control of ribosomal RNA biogenesis.

Laribee RN, Hosni-Ahmed A, Workman JJ, Chen H - PLoS Genet. (2015)

Ccr4-Not binds the rDNA and interacts with RNA Pol I.(A) Schematic of the rDNA locus. The relative position of the qPCR primers used are shown below as black bars. (B) ChIP of Ccr4-TAP, Not1-TAP, and Not2-TAP at the rDNA. The results are the average and standard deviation (SD) of three to four independent experiments. (C) Ccr4 associates with Pol I. 500 μg of cell extract from the indicated strains was utilized for α-HA immunoprecipitation and the presence of Ccr4 determined by α-Myc immunoblot. Statistical significance was determined by Student’s t-test. *- p< 0.05; **- p<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4376722&req=5

pgen.1005113.g001: Ccr4-Not binds the rDNA and interacts with RNA Pol I.(A) Schematic of the rDNA locus. The relative position of the qPCR primers used are shown below as black bars. (B) ChIP of Ccr4-TAP, Not1-TAP, and Not2-TAP at the rDNA. The results are the average and standard deviation (SD) of three to four independent experiments. (C) Ccr4 associates with Pol I. 500 μg of cell extract from the indicated strains was utilized for α-HA immunoprecipitation and the presence of Ccr4 determined by α-Myc immunoblot. Statistical significance was determined by Student’s t-test. *- p< 0.05; **- p<0.01.
Mentions: Specific Ccr4-Not mutants result in significant growth defects which prompted us to ask whether Ccr4-Not might contribute to Pol I transcription since rRNA biogenesis is rate limiting for cell growth and proliferation [35]. Utilizing yeast strains carrying C-terminal TAP epitope tags integrated at either the CCR4, NOT1, or NOT2 genomic loci, we cultured cells in nutrient rich media (YPD) and performed chromatin immunoprecipitation (ChIP) coupled with quantitative PCR utilizing primers to the rDNA locus as outlined (Fig. 1A). All three Ccr4-Not subunits were significantly enriched above background suggesting that Ccr4-Not physically localizes to rDNA repeats (Fig. 1B) [43]. Next, we engineered strains to express either Rpa190-6XHA (Pol I catalytic subunit), Ccr4-13XMyc, or both, and performed co-immunoprecipitation (co-IP) experiments to determine if Ccr4-Not associates with Pol I. Precipitation of Pol I readily co-precipitated Ccr4, suggesting these complexes physically interact (Fig. 1C). Collectively, these data are consistent with the possibility that Ccr4-Not directly regulates Pol I transcription.

Bottom Line: Furthermore, ccr4Δ is synthetically sick when paired with rpa12Δ and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation.Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation.Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis.

View Article: PubMed Central - PubMed

Affiliation: University of Tennessee Health Science Center Department of Pathology and Laboratory Medicine and the UT Center for Cancer Research, Memphis, Tennessee, United States of America.

ABSTRACT
Ribosomal RNA synthesis is controlled by nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 regulates ribosomal RNA expression by affecting RNA Polymerase I (Pol I)-dependent transcription of the ribosomal DNA (rDNA) but the mechanisms involved remain obscure. This study provides evidence that the Ccr4-Not complex, which regulates RNA Polymerase II (Pol II) transcription, also functions downstream of mTORC1 to control Pol I activity. Ccr4-Not localizes to the rDNA and physically associates with the Pol I holoenzyme while Ccr4-Not disruption perturbs rDNA binding of multiple Pol I transcriptional regulators including core factor, the high mobility group protein Hmo1, and the SSU processome. Under nutrient rich conditions, Ccr4-Not suppresses Pol I initiation by regulating interactions with the essential transcription factor Rrn3. Additionally, Ccr4-Not disruption prevents reduced Pol I transcription when mTORC1 is inhibited suggesting Ccr4-Not bridges mTORC1 signaling with Pol I regulation. Analysis of the non-essential Pol I subunits demonstrated that the A34.5 subunit promotes, while the A12.2 and A14 subunits repress, Ccr4-Not interactions with Pol I. Furthermore, ccr4Δ is synthetically sick when paired with rpa12Δ and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation. Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation. Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis.

Show MeSH