Ribosomal RNA synthesis by RNA polymerase I is subject to premature termination of transcription
- University of Toulouse, CNRS, Centre de Biologie Integrative (CBI), France
- Universität Regensburg, Regensburg Center of Biochemistry (RCB), Lehrstuhl Biochemie III, Germany
- BigA, University of Toulouse, CNRS, Centre de Biologie Integrative (CBI), France
- Laboratoire de Physique Théorique de la Matière Condensée (LPTMC), Sorbonne Université, France
Figures
Figure 1
Cells bearing SuperPol overproduce ribosomal RNA (rRNA) in vivo.
(A) Yeast rDNA unit is represented, with the position of the corresponding antisense oligonucleotides used to load slot-blots (see Materials and methods for description). (B) Pol I transcriptional monitoring assay (TMA) was performed in RPA135:F301S and otherwise isogenic wild-type cells grown to mid-log phase in phosphate-depleted YPD medium. Nascent transcripts were labeled with Phosphorus-32 ([32P]) for 40 s. 3’ marked newly synthesized RNAs were extracted, partially hydrolyzed, and revealed using slot-blots. Each labeling was performed independently in triplicate; one representative example is shown in the panel. (C) Results represented in panel B were quantitated. IGS2 and Pol I (5’ETS, 18S.2, 25S.1, and 3’ ETS) are quantified relative to 5S signal in the lower panel. Error bars indicate mean ± SD. ***p<0.005; **p<0.01; *p<0.05, calculated by two-sample t-test.
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Figure 1—source data 1
Original file containing slot Blots for Figure 1B.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig1-data1-v1.zip
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Figure 1—source data 2
Original file containing slot Blots for Figure 1B, indicating the relevant lanes used.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig1-data2-v1.zip
Figure 2 with 1 supplement
SuperPol displays a lower rate of pause compared to wild-type (WT) polymerase I (Pol I).
(A) CRAC distribution profiles obtained for WT Pol I (blue line) and SuperPol (red line). Lines correspond to the mean frequency of reads obtained for three independent experiments. The area of the 35S rDNA gene has been highlighted (5’ETS from +1 to 700 nt; 18S from 701 to 2499 nt; ITS1 from 2500 to 2858 nt; 5.8S from 2859 to 3019 nt; ITS2 from 3020 to 3250 nt; 25S from 3251 to 6647 nt; 3’ETS from 6648 to 6859 nt). (B) Zoom-in 5’ETS CRAC distribution profiles obtained for WT Pol I (blue) and SuperPol (red). Lines correspond to the mean frequency of reads obtained for three independent experiments, while the colored area corresponds to the min-max range of the three experiments. The gray area corresponds to the difference between WT Pol I and SuperPol frequency of reads at each position of the gene. This difference has been calculated using the min-max range values, giving the lowest difference (white area between blue and red min-max range). The lower panel represents the ratio between WT Pol I and SuperPol, represented in log2. Blue areas correspond to sequences where WT is more accumulated than SuperPol and red areas correspond to the sequences where SuperPol is more accumulated than WT Pol I. (C) Frequency of reads obtained for each 35S area (5’ETS, 18S, ITS1, 5.8S, ITS2, 25S, 3’ETS) from three independent experiments was summed and shown as mean ± SD. ***p<0.005; **p<0.01; *p<0.05, calculated by two-sample t-test.
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Figure 2—source data 1
Excel spreadsheet containing the quantitative analysis of the CRAC experiment shown in Figure 2.
The file includes read counts, normalization steps used to generate the profiles presented in the figure.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig2-data1-v1.zip
Figure 2—figure supplement 1
SuperPol displays a lower rate of pause compared to wild-type (WT) polymerase I (Pol I).
(A) CRAC distribution profiles obtained for WT Pol I (blue line) and SuperPol (red line). Lines correspond to the mean frequency of reads obtained for two independent experiments. The area of the 35S rDNA gene has been highlighted. (B) Zoom-in 5’ETS CRAC distribution profiles obtained for WT Pol I (blue) and SuperPol (red). In the upper panel, lines correspond to the mean frequency of reads obtained for two independent experiments, while the colored area corresponds to the min-max range of the two experiments. The gray area corresponds to the difference between WT Pol I and SuperPol frequency of reads at each position of the gene. This difference has been calculated using the min-max range values giving the lowest difference (white area between blue and red min-max range). The lower panel represents the ratio between WT Pol I and SuperPol, represented in log2. Blue areas correspond to sequences where WT is more accumulated than SuperPol and red areas correspond to the sequences where SuperPol is more accumulated than WT Pol I.
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Figure 2—figure supplement 1—source data 1
Excel spreadsheet containing the quantitative analysis of the CRAC experiment shown in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig2-figsupp1-data1-v1.zip
Figure 3
Modified elongation properties of SuperPol impact premature termination of transcription.
(A) Events of premature terminations lead to the releasing of both stalled polymerase I (Pol I) elongation complex and nascent transcript. The 3’end of these abortive transcripts is not protected anymore by the elongation complex and becomes accessible to degradation by rrp6, the 3’ to 5’ exonuclease nuclear subunit of the exosome. To accumulate and detect abortive transcripts, deletion of rrp6 is necessary. (B) Cells bearing wild-type (WT) Pol I or SuperPol, with or without rrp6, were grown, and total RNAs were extracted and analyzed by northern blot using TSS probes. Low-molecular-weight RNA products were resolved on 8% polyacrylamide/8.3 M urea gels. (C) Low-molecular-weight RNA (≈ 80 nt) products were quantified relative to 5S signal. Error bars indicate mean ± SD. ***p<0.005; **p<0.01; *p<0.05, calculated by two-sample t-test. (D) Cumulative distribution function (CDF) of Pol I profiles obtained by CRAC on the 6861 nt of the rDNA. The cumulative distribution of both WT Pol I and SuperPol is represented, respectively, by the blue and red curves. Lines correspond to the mean cumulative distribution of frequency of reads obtained for three independent experiments, while the colored area corresponds to the min-max range of the three experiments.
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Figure 3—source data 1
Original files containing Northern blots for Figure 3B.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig3-data1-v1.zip
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Figure 3—source data 2
Excel spreadsheet containing the annotated files used for Figure 3B and quantitative analysis of triplicates.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig3-data2-v1.zip
Figure 4 with 1 supplement
Purification of RNA polymerase I (Pol I) and SuperPol.
(A) Tagged wild-type (WT) Pol I and SuperPol were immunopurified, respectively, from yeast strains #3207 and #3208. Purified fractions were migrated and separated on 4–12% SDS-PAGE and revealed by Coomassie Blue (see Materials and methods). (B) Tagged WT Pol I and SuperPol were immunopurified, respectively, from yeast strains #3207 and #3208. Purified fractions were analyzed by western blot using anti-Pol I antibody (see Materials and methods).
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Figure 4—source data 1
Original files for Coomassie and Western Blot analysis shown in Figure 4.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig4-data1-v1.zip
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Figure 4—source data 2
Excel spreadsheet containing the annotated files used for Figure 4.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig4-data2-v1.zip
Figure 4—figure supplement 1
Purification of RNA polymerase I (Pol I) and SuperPol and in vitro elongation assay.
(A) Tagged wild-type (WT) Pol I and SuperPol were immunopurified, respectively, from yeast strains #3207 and #3208. Purified fractions from three independent purifications were migrated and separated on 4–12% SDS-PAGE and revealed by Coomassie Blue (see Materials and methods). (B) Tagged WT Pol I and SuperPol were immunopurified, respectively, from yeast strains #3207 and #3208. Purified fractions from three independent purifications were analyzed by western blot using anti-Pol I antibody (see Materials and methods). (C–E) Elongation assay from three independent purifications. About 0.15 pmol of either WT Pol I or SuperPol were added to 0.05 pmol cleavage scaffold and incubated with or without nucleotides (200 μM final concentration of each) for the indicated time intervals. Fluorescent transcripts were analyzed on a 20% denaturing polyacrylamide gel. Uncleaved RNAs (0), RNAs shortened by two (–2) nucleotides and elongated RNAs (from +1 to +14) are indicated with corresponding sequence.
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Figure 4—figure supplement 1—source data 1
Original gels and blots used in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Excel spreadsheet containing the annotated files used for Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig4-figsupp1-data2-v1.zip
Figure 5
Enzymatic properties of SuperPol result in altered cleavage activity and increased elongation activity.
(A) DNA-RNA hybrid scaffold with three mismatched nucleotides at the RNA 3’end used for cleavage assays (clv3). The RNA contains a fluorescent Cy5 label on the 5’RNA end. Cleavage sites are indicated relative to the 3’end of the RNA (site 0). (B) About 0.15 pmol of either wild-type (WT) polymerase I (Pol I) or SuperPol were added to 0.05 pmol cleavage scaffold and incubated for the indicated time intervals. Fluorescent transcripts were analyzed on a 20% denaturing polyacrylamide. RNAs shortened by two (–2) or four (–4) nucleotides and uncleaved RNAs (0) are indicated. (C) Quantification of cleavage efficiency (uncleaved (0) RNA) and the accumulation of –2 and –4 RNA from three independent assays. Values are indicated as a percentage of (0) uncleaved RNA after incubation of scaffold template clv3 with WT Pol I and SuperPol. (D) DNA-RNA hybrid scaffold with one mismatched nucleotide at the RNA 3’end used for elongation assays (clv1). The RNA contains a fluorescent Cy5 label on the 5’RNA end. Cleavage sites are indicated relative to the 3’end of the RNA (site 0). (E) About 0.15 pmol of either WT Pol I or SuperPol was added to 0.05 pmol cleavage scaffold and incubated with or without nucleotides (200 µM final concentration of each) for the indicated time intervals. Fluorescent transcripts were analyzed on a 20% denaturing polyacrylamide gel. Uncleaved RNAs (0), RNAs shortened by two (–2) nucleotides, and elongated RNAs (from +1 to +14) are indicated with corresponding sequence. (F) Quantification of elongation efficiency (+14 nt RNA elongation product) from three independent assays. Values were quantified relative to (0) uncleaved RNA after incubation of scaffold template clv1 with WT Pol I and SuperPol, with or without nucleotides. Error bars indicate mean ± SD. ***p<0.005; **p<0.01; *p<0.05, calculated by two-sample t-test. (G) RNA-DNA hybrid scaffold including no mismatches used for misincorporation assays (ext1). The RNA contains a fluorescent Cy5 label on the 5’RNA end. Ribonucleotides to be incorporated are indicated in gray. (H) About 0.2 pmol Pol I WT, 0.2 pmol SuperPol, and 0.2 pmol Pol I Rpa12ΔC were incubated with 0.066 pmol RNA-DNA scaffold for 20 min on ice. Mixtures of GTP and ATP (200 μmol) were added, and samples were incubated at 28°C for the indicated time intervals. Resulting transcripts were analyzed on a 20% denaturing polyacrylamide gel. RNAs shortened by one (–1) nucleotide, uncleaved RNAs (0), and extended RNAs by one (+1) and two (+2) nucleotides are indicated. Note that production of +2 RNAs reveals misincorporation as the second nucleotide to be incorporated should be UTP and not GTP nor ATP. (I) Quantification of misincorporation from three independent assays. The production of elongated RNA resulting from misincorporation (+2) relative to uncleaved RNA (0) from WT, SuperPol, and Rpa12ΔC was quantified. Note that significant misincorporation of SuperPol relative to WT is detected after a 20 min incubation. Error bars indicate mean ± SD. ***p<0.005; **p<0.01; *p<0.05, calculated by two-sample t-test.
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Figure 5—source data 1
Original gels used in Figure 5B, E and H.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig5-data1-v1.zip
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Figure 5—source data 2
Excel spreadsheet containing the annotated files used for Figure 5B, E and H.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig5-data2-v1.zip
Figure 6
SuperPol is resistant to BMH-21 and retains high levels of transcription upon BMH-21 treatment.
(A) SuperPol suppresses the growth defect due to BMH-21 in the yeast S. cerevisiae. Ten-fold serial dilutions of wild-type (WT) and RPA135-F301S single mutant were spotted on rich media to assess growth at 30°C in the presence of various concentrations of BMH-21 or vehicle. Growth was evaluated after 2 days. (B) Cells bearing SuperPol keep strong polymerase I (Pol I) transcriptional activity upon BMH21 treatment. Pol I transcriptional monitoring assay (TMA) allowing detection of accumulated newly synthesized RNA during the pulse. Nascent transcripts were labeled and detected using antisense oligonucleotides immobilized on slot-blot as described in Figure 1 and Materials and methods. IGS2 and Pol I (mean of 5’ETS, 18S.2, 25S.1, 3’ ETS) are quantified relative to 5S signal in the right panel. Yeast rDNA unit is represented in the upper panel, with the position of the corresponding antisense oligonucleotides used. (C) Cells bearing SuperPol maintained high levels of Rpa190 and Rpa135 upon BMH-21 treatment. WT and RPA135-F301S cells were incubated with 35 µM of BMH21 or DMSO. Aliquots were collected at 15 min, 30 min, and 2 hr, and Pol I subunits’ accumulation was assessed using anti-Pol I antibody (see Materials and methods). (D) Accumulation of the two largest Pol I subunits Rpa190 (left panel) and Rpa135 (right panel) upon BMH-21 treatment is quantified relative to control condition (DMSO). Error bars indicate mean ± SD. ***p<0.005; **p<0.01; *p<0.05, calculated by two-sample t-test.
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Figure 6—source data 1
Original pictures and blots used for Figure 6A-C.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig6-data1-v1.zip
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Figure 6—source data 2
Annotated pictures and blots used for Figure 6A-C.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig6-data2-v1.zip
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Figure 6—source data 3
Excel spreadsheet containing quantitative analysis of triplicates of Figure 6C.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig6-data3-v1.zip
Figure 7
BMH-21 reduces polymerase I (Pol I) occupancy through targeting of paused Pol I and stimulation of premature termination.
(A) Zoom-in 5’ external transcribed spacer (5’ETS) CRAC distribution profiles obtained for wild-type (WT) Pol I (blue) and WT Pol I incubated with 35 µM of BMH-21 for 30 min (green). In the upper panel, lines correspond to the mean frequency of reads obtained for two independent experiments, while the colored area corresponds to the min-max range of the two experiments. The gray area corresponds to the difference of frequency of reads at each position of the gene between WT Pol I and WT Pol I with BMH-21. This difference has been calculated using the min-max range values, giving the lowest difference (white area between blue and red min-max range). The lower panel represents the ratio between WT Pol I and WT Pol I with BMH-21, plotted on a log2 scale. Blue areas correspond to sequences where WT Pol I without BMH-21 is more accumulated, and green areas correspond to the sequences where WT Pol I incubated with BMH-21 is more accumulated. (B) Zoom-in 5’ETS CRAC distribution profiles obtained for SuperPol (Red) and SuperPol incubated with 35 µM of BMH-21 for 30 min (yellow). (C) Cells bearing WT Pol I or SuperPol, in the presence or absence of Rrp6, were grown and treated with 35 µM of BMH-21 for 30 min. Total RNAs were extracted and analyzed by northern blot using transcription start site (TSS) probe. Abortive transcripts are indicated by arrows. (D) Premature transcript products were quantified relative to 5S signal (focusing on ≈80 nt size). Error bars indicate mean ± SD. ***p<0.005; **p<0.01; *p<0.05, calculated by two-sample t-test.
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Figure 7—source data 1
Original files used for Figure 7C.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig7-data1-v1.zip
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Figure 7—source data 2
Annotated Northern blots used for Figure 7C.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig7-data2-v1.zip
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Figure 7—source data 3
Excel spreadsheet containing the quantitative analysis of the CRAC experiment shown in Figure 7.
- https://cdn.elifesciences.org/articles/106503/elife-106503-fig7-data3-v1.zip
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Ribosomal RNA synthesis by RNA polymerase I is subject to premature termination of transcription
eLife 14:RP106503.
https://doi.org/10.7554/eLife.106503.3
