Cells bearing SuperPol overproduce rRNA in vivo

Figure 1A: Yeast rDNA unit is represented, with the position of the corresponding antisense oligonucleotides used to load Slot blots (see Materials and methods for description).

Figure 1B: Pol I 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 labelled with Phosphorus-32 ([32P]) for 40 seconds. 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.

Figure 1C: 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.

SuperPol displays a lower rate of pause compared to WT Pol I

Figure 2A: 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 700nt ; 18S from 701 to 2499nt ; ITS1 from 2500 to 2858nt ; 5.8S from 2859 to 3019nt; ITS2 from 3020 to 3250nt; 25S from 3251 to 6647nt; 3’ETS from 6648 to 6859nt).

Figure 2B: 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 grey 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.

Figure 2C: Frequency of reads obtained for each 35S area (5’ETS, 18S, ITS1, 5,8S, ITS2, 25S, 3’ETS) from three independent experiments were summed and shown as mean+/- SD. *** P < 0,005 ; ** P < 0 . 01 ;* P < 0 . 05, calculated by two-sample t -test.

Modified elongation properties of SuperPol impact premature termination of transcription.

Figure 3A: Events of premature terminations lead to the releasing of both stalled Pol I elongation complex and nascent transcript. The 3’end of these abortive transcripts is not protected anymore by the elongation complex and become 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. Figure 3B: Cells bearing WT Pol I or SuperPol, with or without rrp6, were grown and total RNAs were extracted and analyzed by northernblot using TSS probes. Low molecular weight RNA products were resolved on 8% Polyacrylamide/8.3M urea gels.

Figure 3C: Low molecular weight RNA (≈ 80nt) 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. Figure 3D: Cumulative Distribution Function (CDF) of Pol I profiles obtained by CRAC on the 6861 nucleotides of the rDNA. The cumulative distribution of both WT Pol I and SuperPol are 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.

Purification of RNA Pol I and SuperPol

Figure 4A: Tagged 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).

Figure 4B: 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).

Enzymatic properties of SuperPol result in altered cleavage activity and increased elongation activity

Figure 5A: 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 relatively to the 3’end of the RNA (site 0).

Figure 5B: About 0.15 pmol of either WT 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.

Figure 5C: Quantification of cleavage efficiency (uncleaved (0) RNA) and the accumulation of -2 and -4 RNA from three independent assays. Values are indicated as percentage of (0) uncleaved RNA after incubation of scaffold template clv3 with WT Pol I and SuperPol.

Figure 5D: 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 relatively to the 3’end of the RNA (site 0).

Figure 5E: 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) and are indicated with corresponding sequence.

Figure 5F: Quantification of elongation efficiency (+14nt 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.

SuperPol is resistant to BMH-21 and retains high levels of transcription upon BMH21 treatment.

Figure 6A: SuperPol suppresses the growth defect due to BMH-21 in the yeast S. cerevisiae. Ten-fold serial dilutions WT and RPA135-F301S single mutant were spotted on rich media to assess growth at 30°C in presence of various concentration of BMH-21 or vehicle. Growth was evaluated after two days. Figure 6B: Cells bearing SuperPol keep strong Pol I transcriptional activity upon BMH21 treatment. Pol I TMA allowing detection of accumulated newly synthesized RNA during the pulse. Nascent transcripts were labelled, and revealed using antisens oligonucleotides immobilized on slot-blot as described in Figure 1 and Materials and Methods. IGS2, 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.

Figure 6C: Cells bearing SuperPol maintained high levels of Rpa190 and Rpa135 upon BMH-21 treatment. WT and RPA135-F301S cells were incubated with 35µM of BMH21 or DMSO. Aliquots were collected at 15 minutes, 30 minutes and 2 hours and Pol I subunits accumulation was assessed using anti-Pol I antibody (see Materials and Methods).

Figure 6D: Accumulation of the two largest Pol I subuntis 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.

BMH-21 reduces Pol I occupancy through targeting of paused Pol I and stimulation of premature termination

Figure 7A: Zoom in 5’ETS CRAC distribution profiles obtained for WT Pol I (Blue) and WT Pol I incubated with 35µM of BMH-21 for 30 minutes (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 grey 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, represented in log2. 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.

Figure 7B: Zoom in 5’ETS CRAC distribution profiles obtained for SuperPol (Red) and SuperPol incubated with 35µM of BMH-21 for 30 minutes (Yellow).

Figure 7C: Cells bearing WT Pol I or SuperPol, in presence or in absence of Rrp6, were grown and treated with 35µM of BMH-21 for 30 min. Total RNAs were extracted and analyzed by northern blot using TSS probe. Abortive transcripts are indicated by arrows.

Figure 7D: 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.

SuperPol displays a lower rate of pause compared to WT Pol I

Figure S1A: 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.

Figure S1B: 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 minmax range of the two experiments. The grey 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.

Purification of RNA Pol I and SuperPol and in vitro elongation assay

Figure S2A: Tagged WT Pol I and SuperPol were immunopurified respectively from yeast strains #3207 and #3208. Purified fractions from three independant purifications were migrated and separated on 4-12% SDS-PAGE and revealed by Coomassie Blue (see Materials and Methods).

Figure S2B: Tagged WT Pol I and SuperPol were immunopurified respectively from yeast strains #3207 and #3208. Purified fractions from three independant purifications were analyzed by western blot using anti-Pol I antibody (see Materials and Methods).

Figure S2C ; S2D; S2E : Elongation assay from three independant 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) and are indicated with corresponding sequence.