4sUDRB-seq: measuring genomewide transcriptional elongation rates and initiation frequencies within cells

Abstract

Although transcriptional elongation by RNA polymerase II is coupled with many RNA-related processes, genomewide elongation rates remain unknown. We describe a method, called 4sUDRB-seq, based on reversible inhibition of transcription elongation coupled with tagging newly transcribed RNA with 4-thiouridine and high throughput sequencing to measure simultaneously with high confidence genome-wide transcription elongation rates in cells. We find that most genes are transcribed at about 3.5 Kb/min, with elongation rates varying between 2 Kb/min and 6 Kb/min. 4sUDRB-seq can facilitate genomewide exploration of the involvement of specific elongation factors in transcription and the contribution of deregulated transcription elongation to various pathologies.

Figure 1

Adaptation of the DRB assay for measuring elongation rates in short time scales by RNA sequencing. (A) Analysis of OPA1 and TTC17 pre-RNA in HeLa cells, immediately after 3 h DRB treatment (DRB) and at the indicated time points after DRB removal. NT = non-treated. Pre-mRNA levels were determined by qRT-PCR, employing intronic primers. All values were normalized to 18S RNA in the same sample. Levels in non treated cells were set as 1. Bars indicate averages of three independent experiments; error bars represent standard deviation. (B) Relative pre-mRNA/mRNA ratios in total RNA (CON) and 4sU-enriched RNA (4sU). HeLa cells were labeled for 8 min with 4sU (1mM). RNA was isolated, biotinylated, and enriched on streptavidin beads. qRT-PCR analysis of OPA1 and TTC17 pre-mRNA and mRNA was performed on the enriched RNA, as well as on total (non-enriched) RNA. The pre-mRNA/mRNA ratio for each gene in the CON sample was arbitrarily defined as 1.0. (C) Scheme of the timing of DRB and 4sU treatments in the different conditions. NT = non-treated, DRB = 3 h of DRB only, 4 min = 3 h of DRB and harvesting 4 min after DRB removal, 8 min = 3 h of DRB and harvesting 8 min after DRB removal. Red squares = 4sU, orange stars = biotin. Following the indicated treatments, isolated 4sU-labeled RNA was biotinylated and purified with magnetic streptavidin beads as described in [42] and subjected to high throughput sequencing.

Figure 2

In vivo elongation rate analysis of thousands of genes simultaneously. (A) Averaged distribution of 4sUDRB-seq reads over the different biological repeats in all genes longer than 20 Kb in RNA harvested 4 or 8 min after DRB removal. To minimize distortion of the data by residual mature mRNA, only reads within introns were considered and averaged. (B, C). 4sUDRB-seq reads distribution along the RBBP6 and PFDN1 genes. Arrow marks the direction of transcription.

Figure 3

In vivo genomewide measurement of transcription elongation rates. (A) Schematic representation of the algorithm used to infer elongation rates, exemplified for the NDUFV2 gene. Left panel: 4sUDRB-seq data for 0/4/8 min after DRB release (similar to Figure 2B). Each signal was then corrected for the inferred background signal (right top panel), identifying for the 4 and 8 min samples the first position downstream to the TSS where the corrected signal is similar to that of the 0 time point; this location is designated by a vertical dashed line. To refine the boundary identification, we evaluated the background-corrected signals of the 4 and 8 min time points divided by the 0 time point signals and their derivatives. Refinement of the boundary position was then performed by monitoring the decreasing signal area in the vicinity of the rough boundary estimate, and determining the location between the closest peak and the point where the signal plateaus. (B) Linear fitting was performed on the averaged 4 and 8 min elongation boundaries as a function of time for the indicated genes. Elongation rates were defined by extracting the slope value of the linear fit (V). Confidence interval is indicated for each gene. (C) Distribution of measured elongation rates of all informative DRB-sensitive genes. (D) Box-whisker plot of log2 transformed H3K36me3 and H3K79me2 levels in genes that overlap with each modification in the 25% of genes with the highest calculated elongation rate and the 25% of genes with the lowest elongation rate. H3K79me2 is significantly higher in fast elongating genes (t-test, P = 1e-7). All data were adapted from ENCODE.

Figure 4

Calculation of genomewide relative transcription initiation frequencies by 4sUDRB-seq. (A) Schematic representation of the linear decrease in 4sUDRB-seq read intensity downstream to the TSS of a hypothetical gene and in a gene with similar elongation rate but higher initiation frequency (“Higher Frequency”) or in a gene with similar initiation frequency but faster elongation rate (“Faster Elongation”). (B) Analysis as in (A), exemplified for the 8 min time point of the SRFBP1 gene, Red: linear fit of the signal upstream to the boundary found by our algorithm. (C) Average expression levels of four quartiles of genes binned by increasing initiation frequencies. r indicates Pearson correlation. Error bars = standard errors.