Global analysis of short RNAs reveals widespread promoter-proximal stalling and arrest of Pol II in Drosophila

Abstract

Emerging evidence indicates that gene expression in higher organisms is regulated by RNA polymerase II stalling during early transcription elongation. To probe the mechanisms responsible for this regulation, we developed methods to isolate and characterize short RNAs derived from stalled RNA polymerase II in Drosophila cells. Significant levels of these short RNAs were generated from more than one-third of all genes, indicating that promoter-proximal stalling is a general feature of early polymerase elongation. Nucleotide composition of the initially transcribed sequence played an important role in promoting transcriptional stalling by rendering polymerase elongation complexes highly susceptible to backtracking and arrest. These results indicate that the intrinsic efficiency of early elongation can greatly affect gene expression.

Fig. 1. Short capped RNAs are produced by promoter-proximal Pol II

Distribution of RNA 5’-ends (blue) and Pol II ChIP-seq signal (red) on (A) myoglianin (CG1838) a gene with stalled Pol II and low expression and (B) CG10289, a gene with more uniform polymerase distribution and high expression. The peak number of RNA reads (1-nt windows) and Pol II ChIP-seq reads (25-bp windows) is indicated in the corresponding color. Insets contain magnified views of the TSSs. (C) The number of short RNAs that mapped to stalled genes (median reads/TSS=1,477) as compared to genes that were not considered stalled (median reads/TSS=178). Box plots represent the 25^th, 50^th and 75^th percentiles, with whiskers denoting the 5^th and 95^th percentiles. (D) Metagene analysis of 5’-end reads aligned to observed TSSs.

Fig. 2. 3’-ends of short RNAs identify sites of polymerase stalling

5’-end (blue) and 3’-end (orange) RNA reads are shown for example genes, designated by Flybase gene names, with observed TSSs indicated by arrows. The number of 5’- and 3’-end reads at the peak locations is given in the corresponding colors. Permanganate footprints from nuclei are shown for each gene, alongside purified DNA and the A+G DNA ladder. Numbers in black indicate positions relative to the TSS, and brackets show areas of permanganate reactivity.

Fig. 3. Promoter-proximal sequences impact Pol II stalling

(A) 3’-end reads were aligned relative to observed TSSs. RNAs shorter than 26 nt are not efficiently mapped to the genome (colored in yellow). (B) Metagene analysis of melting temperatures (°C) for 9-bp sequences across the initially transcribed regions for genes that generate significant numbers of short RNAs (orange) and all other genes (black). (C) Tm analysis of 9-bp sequences surrounding the primary 3’-end location for a subset of genes with focused 3’-end positions (N=434).

Fig. 4. Stalled polymerase complexes are predominantly backtracked

(A) The role of transcription cleavage factor IIS in rescuing arrested Pol II elongation complexes. Following transcriptional pausing (top panel) the polymerase can backtrack along DNA (middle panel), which dislodges the RNA 3’-end from the polymerase active site (shown as red dot) and blocks further transcription. IIS induces internal cleavage of nascent RNA (bottom panel), re-aligning the 3’-end with the active site, such that Pol II can resume transcription. (B) Depletion of IIS leads to an increase in RNA length within the promoter-proximal region. Shown is the difference between the normalized number of reads in IIS-depleted and mock-treated samples, binned in 5-nt windows. (C) IIS-depletion affects RNA 3’-end positions but not permanganate reactivity profiles. RNA 3’-ends from mock-treated samples are shown in orange and IIS-depleted cells in green. Brackets denote regions of permanganate reactivity.