High-resolution genome-wide maps of transcribing RNAPII in strains expressing different Sen1 variants.Figure 4RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. A) Metagene analysis of the RNAPII distribution relative to the annotated polyadenylation site (PAS) of mRNAs. Values on the y axis correspond to the median coverage. B) Box-plot representation of the readthrough (RT) index as a readout of the termination efficiency at each mRNA. The RT index was calculated as the average signal over a window of 200 nt downstream of the PAS, divided by the average signal from the TSS to the PAS of each mRNA. Boxes include all the values between the 25th and the 75th percentiles and the horizontal line indicates the median. The upper error bars indicate the distance between the largest value smaller than or equal to the upper limit of the box + 1.5 *IQR (where IQR is the interquartile distance or distance between the first and the third quartile). The lower error bars indicate the distance between the lowest value greater than or equal to the lower limit of the box - 1.5 *IQR. For each mRNA (n=5752), the values correspond to the average of two independent biological replicates. Data populations were compared using a t-test (bilateral distributions, unpaired data) and p-values≥ 0.05 are indicated as ns (not significant). RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. C-D) IGV screenshots of two well-characterized protein-coding genes displaying substantial defects in premature termination in some mutants. The values correspond to the counts per million of counts (CPM) multiplied by 10. RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. E) Metagene analyses of transcribing RNAPII at CUTs performed as for mRNAs but using the annotated transcription termination site (TTS) as the reference point. F) Analysis of termination defects in the different mutants at CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 400 nt downstream of the TTS, divided by the average signal over a window of 100 nt downstream from the TSS (CUT100). For each CUT (n=329), the values correspond to the average of two independent biological replicates. P-values < 0.001 are indicated by three asterisks. RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. G-H) IGV screenshots of examples of CUTs displaying substantial termination defects in the mutants. RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. I) Venn diagrams illustrating the overlap between the CUTs that display statistically significant termination defects (p-value≤ 0.05, see methods for details) in the different mutants. The size of the circles is not proportional to the number of CUTs. RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs. Box-plots performed as in (B). The RT index is calculated as the average signal over a window of 300 nt downstream of the TTS, divided by the average signal over the mature snoRNA. RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Analysis of RNAPII occupancy around snoRNAs performed as for CUTs. RNAPII CRAC were performed in the presence of the indicated versions of SEN1 expressed from its own promoter in a centromeric plasmid, upon depletion of the chromosomally-encoded version of Sen1 in a Sen1-AID strain. Venn diagrams illustrating the overlap between the CUTs that display statistically significant termination defects (p-value≤ 0.05, see methods for details) in the different mutants.