Global profiling of AS and mRNA levels affected by DRB and camptothecin inhibition of RNA polymerase II elongation. (A) Overview of the experimental and data analysis strategy. Jurkat T cells were mock-treated (with DMSO) or treated with three different concentrations of DRB (10, 25, and 50 μg/mL in DMSO) or camptothecin (4, 8, and 16 ng/mL in DMSO) for 24 h, and then stimulated with PMA for 6 h. Cy3- and Cy5-labeled cDNA prepared from poly(A)⁺ RNA isolated from the treated cells was hybridized to a custom microarray containing sets of exon and junction-specific probes (bold lines) representing 5782 human cassette-type AS events that are conserved in mouse transcripts. A, alternative exon; C1 and C2, 5′ and 3′ flanking constitutive exons. (B) Analysis of the microarray data reveals genes with changes in AS and mRNA levels. The Venn diagrams indicate the numbers of genes that change in each direction (color-coded according to increased/decreased mRNA level and increased/decreased splicing level) upon DRB and camptothecin (Camp) treatments. Numbers of genes with significantly overlapping changes at the mRNA and splicing levels are indicated in red (P-value < 7.0 × 10⁻⁵). Only genes with coincident increased exon inclusion and decreased mRNA levels are significantly enriched in Gene Ontology (GO) categories (red text; FDR < 0.005).

Validation of microarray-detected changes in AS and mRNA levels following Pol II elongation inhibition. (A,B) Primer pairs specific for constitutive exons flanking each alternative exon (see Fig. 1A) were used to amplify exon-included and exon-skipped spliced variants, which are indicated by red and yellow circles, respectively. Each RT-PCR analysis was performed in triplicate on total RNA isolated from mock-treated control (“C”) cells, or cells treated with the three different concentrations of DRB (A) or camptothecin (B) (see Fig. 1A). The common color scale represents the percent of transcripts with exon inclusion (%In) and mRNA levels (Trans), as predicted from the microarray data and quantified from the RT-PCR data. Transcript levels on the arcsinh scale were represented as the level in each treatment over the median level for all treatments for the same gene. Numerical values below each panel represent average measurements from the three independent RT-PCR experiments. RT-PCR assays monitoring levels of 5S rRNA and U1 snRNA were used to control for loading and recovery. Additional RT-PCR validation experiments for the same treatments, together with numerical quantification of the data, are shown in Supplemental Figures S2, S3, and S4.

Mutations of Pol II that affect transcription elongation consistently affect splicing and mRNA levels of RNA binding protein genes. (A) RT-PCR assays performed on total RNA isolated from Raji B cell lines expressing tetracycline-inducible, alpha-amanitin-resistant, and HA-tagged RNA Pol IIs harboring alanine-substituting mutations at every Ser 2, Ser 5, or Ser 7 position within the CTD heptapeptide repeats (see Results). The cell lines were treated with tetracycline to induce expression of the alpha-amanitin-resistant polymerases for 24 h, then endogenous Pol II was destroyed by treatment with alpha-amanitin for 48 h. Lysates from equal numbers of cells for each cell line were analyzed for Pol II levels by Western blotting using anti-HA antibody (Anti-HA WB) and total RNA was analyzed by RT-PCR assays as described in Figure 2. Numerical values below each panel represent average measurements from three independent experiments. Additional RT-PCR experiments and numerical quantification of the data are shown in Supplemental Figure S6. (B) Control (Ctrl) and expression plasmids for alpha-amanitin-resistant wild-type (WT) or C4 slow mutant (C4) polymerases were transiently expressed in 293T cells as described in Methods. RNA harvested from the transfected cells was analyzed by RT-PCR as described above. Expression levels of the exogenous Pol IIs were confirmed by Western blotting using an antibody (N-20), which recognizes the N-terminal domain of the largest subunit of Pol II. Tubulin detection was used as loading control for the Western blot, and 5S rRNA detection was used as loading control for RT-PCR assays. Numerical values below each panel represent average measurements from three independent experiments. Additional RT-PCR experiments and numerical quantification of the data are shown in Supplemental Figure S7.

Roles for NMD and cell stress in the control of splice variant levels regulated by Pol II elongation rate. (A) Cycloheximide (CHX) treatment of PMA-stimulated Jurkat cells promotes increased levels of PTC-containing splice variants that also respond to DRB and camptothecin treatments. Jurkat cells were treated with DRB for 24 h, followed by PMA stimulation. Cycloheximide was added an hour after the addition of PMA, and RNA was isolated 5 h later. RT-PCR analysis of the RNA samples was performed as described in Figure 2. Numerical values below each panel represent average measurements from three independent experiments. Additional RT-PCR experiments and numerical quantification of the data are shown in Supplemental Figure S8. (B) Knockdown of the essential NMD factor UPF1 in HeLa cells results in specific increases in exon-included, PTC-containing splice variants that are responsive to treatments that inhibit Pol II. UPF1 was knocked down in HeLa cells by transfection of a specific siRNA (Kim et al. 2005) for 48 h, prior to harvesting total RNA for analysis by RT-PCR assays, which were performed as described in Figure 2. Numerical values below each panel represent average measurements from three independent experiments. Additional RT-PCR experiments and numerical quantification of the data are shown in Supplemental Figure S9. (C) UV irradiation of Jurkat cells results in increased levels of exon inclusion and decreased levels of mRNA for RNA processing factor genes that respond to Pol II elongation perturbation. Jurkat cells were exposed to 120 J/m² UV and allowed to recover for 6 h prior to harvesting total RNA for RT-PCR assays, which were performed as described in Figure 2. Numerical values below each panel represent average measurements from three independent experiments. Additional RT-PCR experiments and numerical quantification of the data are shown in Supplemental Figure S10. Loading/recovery controls in A–C are as shown in Figures 2 and 3.

Increased Pol II occupancy over intron sequences flanking alternative exons that display Pol II elongation rate-dependent regulation of AS. Jurkat cells were treated with or without DRB as described in Figure 1, cross-linked with formaldehyde, and Pol II cross-linked to sheared chromatin fragments was immunoprecipitated with a monoclonal antibody (4H8) specific for the Pol II CTD. Illumina short (44 nt) sequence reads were generated from the immunoprecipitated chromatin fragments and these were mapped to the exons and flanking 500 nt intron sequences, corresponding to the C1–A–C2 regions analyzed by microarray profiling in Figure 1. C1, first/upstream constitutive exon; C2, second/downstream constitutive exon; A, alternative exon; I1, first/upstream intron; I2, second/downstream intron. (A) Plots showing merged distributions of Pol II over the profiled exons and intron sequences of all microarray profiled genes. The Pol II distributions are shown as averages of ratios of read counts at each position over a value representing the total number of reads mapped to all exons and introns in the gene set over the total length of the exons and introns in that set. Lengths of exons were normalized by binning into 100 equal-size segments to facilitate direct comparison. Plots are shown for mock (control) treatment and treatments with 10 and 25 μg/mL DRB. (B) Pol II occupancy for microarray-profiled genes with DRB (25 μg/mL)-dependent increases in exon inclusion and decreases in mRNA levels, designated as “Regulated” genes (red trace). A similar number of genes with comparable mRNA levels but that do not detectably respond to DRB (25 μg/mL) are shown for comparison, designated as “Control” genes (blue trace). Additional control traces are shown in Supplemental Figure S11. The traces show the average ratios of read counts at each position over a sliding window of 20 nt in the DRB-treated versus mock-treated samples (see Methods for details). (C) Bar graph quantifying average ratios of Pol II occupancy within each indicated region, as determined from the data in panel B. Regions displaying significant increases in Pol II occupancy in the DRB-treated relative to mock treated samples, between the Regulated and Control sets of genes, are indicated. Error bars indicate standard error and P-values were calculated using Wilcoxon signed-rank test.

Transcription elongation-dependent regulation of splicing factor genes during starvation of C. elegans. (A) Analysis of the changes in average Pol II distribution (as measured by ChIP-seq) in C. elegans under starvation and growth recovery conditions, for C1–I1–A–I2–C2 regions from a set of splicing factor genes, and C1–I1–A–I2–C2 regions from an equivalent number of randomly selected genes that have comparable expression levels. Bar graphs show the percent change of Pol II signal within each individual region under starvation versus recovery conditions. Analyzed data are from Baugh et al. (2009); see Methods for details. Asterisks highlight statistically significant changes in Pol II occupancy between starvation and recovery conditions (P-values ≤ 1.1 × 10⁻⁴, Wilcoxon signed-rank test). (B) RT-PCR analysis of AS events in splicing factor genes corresponding to those displaying increased Pol II occupancy in flanking intron regions upon starvation conditions (A). RT-PCR assays were performed as described in Figure 2 on total RNA isolated from worms subjected to starvation and recovery conditions. RNA levels were normalized to gdp-2 mRNA. S, starved worms; S+R, starved worms reintroduced to food. Numerical values below each panel represent average measurements from three independent experiments. Additional RT-PCR experiments and numerical quantification of the data are shown in Supplemental Figure S12.