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 over 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.

dDsk2 regulates H2Bub1 and RNA polymerase II pausing at dHP1c complex target genes

“…Polyclonal rat aWOC (IF: 1/500) and rabbit aROW (WB: 1/10,000, ChIP/IP: 1 ml) antibodies were raised against recombinant proteins aa230-626 and aa588-956, respectively. Other antibodies used are described in the indicated references: rat aHP1c (WB: 1/10,000, IF: 1/500, ChIP/IP: 1 ml), aHP1b (WB: 1/ 10,000, ChIP/IP: 1 ml) and aROW (WB: 1/10,000, IF: 1/400, ChIP/IP: 1 ml) 18 , rabbit aWOC (WB: 1/10,000, ChIP/IP: 1 ml) 62 , rabbit and rat aDDP1 (ChIP/IP: 1 ml) 63 , rabbit adDsk2 (WB: 1/12,000, IF: 1/1,000, ChIP/IP: 1 ml) 16 , mouse ap54 (WB: 1/50) 64 , rabbit aNELF-E (WB: 1/500, ChIP/IP: 3 ml) 65 and rabbit aRPB3 (WB: 1/1,500, ChIP: 15 ml) 26 . Commercially available antibodies used were as follows: rabbit aH3K4me3 (Abcam, ab8580, ChIP: 1 ml), rabbit aIIoser5 (Abcam, ab5131, ChIP: 3 ml), rabbit aBre1 (NOVUS, 40280002, WB: 1/1,000, ChIP: 2 ml) and aRad6 (SANTA CRUZ, sc-30078, WB: 1/500, ChIP: 2 ml), mouse aGST (glutathione S-transferase; Novagen, 71097-3, WB: 1/10,000), mouse aGFP (Roche, 1814460, IF: 1/50), mouse aH2Bub1 (Millipore, 05-1312, WB: 1/4,000, ChIP/IP: 1 ml), mouse aTubulin (Millipore, MAB3408, WB: 1/2,000), rabbit aActin (Sigma, A2066, WB: 1/10,000), mouse aV5 (Invitrogen, 460705, WB: 1/5,000), mouse aUbiquitin (SANTA CRUZ, sc-8017, WB: 1/500), rabbit aPanAc (Abcam, Ab193-100, ChIP: 1 ml) and rabbit aH4Ac (Millipore, no.…”

“…shortRNA-Seq data were downloaded from NCBI GEO (GSE18643) 26 . shortRNA sites were defined as those with a score above the third quartile and annotated for overlapping genes as described above.…”

“…P-TEFb also regulates transition of Pol IIo ser5 into the elongating Pol IIo ser2 form, as it phosphorylates Ser2 in the CTD. Pausing of RNA pol II is accompanied by Pol IIo ser5 enrichment at promoters and results in the production of short RNAs 26 and increased reactivity with KMnO 4 , which reacts with unpaired thymines (T) and detects melted DNA at the sites of pausing 27 .…”

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

“…In addition, Ser7 of the CTD can also be phosphorylated, which has been linked to small nuclear RNAs (snRNA) transcription and recruitment of the integrator complex [9][10][11][12][13] . Whereas some genes, for example, those on CpG islands, can directly proceed to the elongation state by recruiting P-TEFb to RNAPII 14 , genomewide studies suggest that the majority of genes in higher eukaryotes are under the control of promoter-proximal pausing [15][16][17] .…”

Enforcing the pause: Transcription factor Sp3 limits productive elongation by RNA polymerase II

“…The kinase activity of P-TEFb Drosophila cells revealed that loss of NELF-mediated pausing reduced RNA Pol II promoter occupancy at most, but not all, promoters, 2 globally implicating NELF in inhibition of early elongation. The high sensitivity of the global runon-sequencing (GRO-seq) technique, 51 allowed the determination that even active genes undergo transient pausing of RNA Pol II, 47,52 suggesting that NELF-mediated pausing may be a general feature of the transcription mechanism. Furthermore, these studies suggest that the efficiency of pause release, rather than the establishment of pausing, plays a major role in determining gene expression levels.…”