Figure 3. Polymerase II (Pol II) density is increased around transcription start sites (TSS), polyadenylation sites, and splice sites (SS).

(A) Metagene plot of normalized mean Pol II occupancy and the surrounding 95% confidence interval for the 500 bp surrounding the most abundant annotated TSS (Pelechano et al., 2013) (n=2415 genes). Metagene for dst1∆ (green) can be compared to the Pol II density in the wild-type strain (gray). (B) Normalized mean Pol II occupancy and the surrounding 95% confidence interval for the 600 bp surrounding the most abundant annotated poly(A) sites (Pelechano et al., 2013) in the antisense orientation. Metagene for dst1∆ (blue) can be compared to the Pol II density in the wild-type strain (gray). (C) Normalized mean Pol II occupancy and the surrounding 95% confidence interval for the 500 bp surrounding the most abundant annotated poly(A) sites (Pelechano et al., 2013). Metagenes for subunits of the Ccr4-NOT complex deleted (red) can be compared to the Pol II density in the wild-type strain (gray). (D) Same as (C), for rtt103∆. (E–F) Normalized mean Pol II occupancy and the surrounding 95% confidence interval for the 50 bp surrounding annotated 5’ and 3’ splice sites (SS). Metagenes for subunits of the Caf1 complex deleted (blue) can be compared to the Pol II density in the wild-type strain (gray). (G) Cartoon and equation illustrating pausing index (PI) calculation. (H) PI for the TSS (green), polyadenylation [poly(A)] (red), and 3’ antisense (blue) regions across genes. Horizontal axis is hierarchically clustered, revealing TSS, poly(A), and antisense pausing indices for genes in wild-type yeast. (I) Same as (H), for 5’ and 3’ SS pausing indices. (J) Scatter plot of the median pausing indices in the TSS and 3’ antisense regions for all deletion strains. Relationship was quantified using Pearson correlation. (K) Same as in (J), comparing pausing the 5’ and 3’ SS surrounding introns. (L) Boxplot of TSS PI distributions in each deletion strain, ordered by median PI. Horizontal solid line indicates median value for wild-type yeast; dotted lines indicate the 45th and 55th percentile of wild-type PI values. (M–P) Same as (L), for 3’ antisense PI, poly(A) site PI, 5’ SS PI, and 3’ SS PI.

Figure 3—figure supplement 1. Heatmaps of polymerase II (Pol II) density around RNA processing sites reveal differences in polymerase behavior across deletion strains, which can have functional consequences in specific deletion strains.

(A) Normalized mean Pol II occupancy and the surrounding 95% confidence interval for –100 to +600 bp surrounding the most abundant annotated transcription start sites (TSS; Pelechano et al., 2013) (n=2415 genes). Metagenes for each deletion strain (green) can be compared to the Pol II density in wild-type strains (gray). Deletion strains are ordered by median pausing index for the TSS region, as in Figure 3F. (B) Normalized mean Pol II occupancy and the surrounding 95% confidence interval for –200 to +500 bp surrounding the most abundant annotated poly(A) sites (Pelechano et al., 2013) (n=2415 genes). Metagenes for each deletion strain (blue) can be compared to the Pol II density in wild-type strains (gray). Deletion strains are ordered by median pausing index for the antisense region, as in Figure 3G. (C) Normalized mean Pol II occupancy and the surrounding 95% confidence interval for the –500 to +200 bp surrounding the most abundant annotated poly(A) sites (Pelechano et al., 2013) (n=2415 genes). Metagenes for each deletion strain (red) can be compared to the Pol II density in wild-type strains (gray). Deletion strains are ordered by median pausing index for the poly(A) region, as in Figure 3H. (D) Normalized mean Pol II occupancy and the surrounding 95% confidence interval for the 50 bp surrounding annotated 5’ (dark blue) and 3’ (light blue) splice sites (SS). Metagenes for each deletion strain can be compared to the Pol II density in wild-type strains (gray) (n=252 genes). Deletion strains are ordered by median pausing index for the 5’ SS region, as in Figure 3I. (E) Cartoon illustrating splicing index calculation. (F) Boxplot showing the distribution of splicing indices calculated in both the cac2∆ and wild-type strain. Significance was determined with a Student’s t-test. RNA-seq data was obtained from Hewawasam et al., 2018.

Figure 3—figure supplement 2. Polymerase II density is increased around RNA processing sites to varying degrees across deletion strains.

(A) Scatterplot of the pausing index (PI) in the transcription start site (TSS) and poly(A) region (top left), TSS and 3’ antisense (top right), poly(A) and 3’ antisense (bottom left), and the 5’ and 3’ splice sites (SS) surrounding introns (bottom right) for each gene in the wild-type strain. The lack of any relationship between these values is quantified by Pearson correlation. (B) Cumulative density plot illustrating the distribution of pausing indices for TSS (green), poly(A) site (red), 3’ antisense (blue), 5’ SS (dark blue), and 3’ SS (light blue) regions. In wild-type yeast, 25% of genes have a TSS PI ≥2.74; this PI value falls to 0.78 for poly(A) PI, 2.51 for 3’ antisense, and 2.18 and 2.35 for 5’ and 3’ SS regions, respectively. Distributions of both SS pausing indices are statistically the same, as determined by a Kolmogorov-Smirnov test (p=0.273). (C) Scatter plot of the median pausing indices in the TSS and poly(A) regions (top) and poly(A) and 3’ antisense (bottom) for all deletion strains, colored as in Figure 1B. Relationship was quantified using Pearson correlation. (D) PI for the TSS region across all non-overlapping protein-coding genes (n=3341). Both axes are hierarchically clustered, revealing genes with similar pausing densities as well as deletion strains that share pausing indices across their genomes. (E–H) Same as in (D), for pausing indices calculated across different gene regions - 3’ antisense, poly(A) sites, 5’ SS, and 3’ SS, respectively.