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. 2011 Jun 28;108(26):10460-5.
doi: 10.1073/pnas.1106630108. Epub 2011 Jun 13.

Antisense RNA polymerase II divergent transcripts are P-TEFb dependent and substrates for the RNA exosome

Ryan A Flynn  1 , Albert E Almada, Jesse R Zamudio, Phillip A Sharp
Affiliations

Antisense RNA polymerase II divergent transcripts are P-TEFb dependent and substrates for the RNA exosome

Ryan A Flynn et al. Proc Natl Acad Sci U S A. .

Abstract

Divergent transcription occurs at the majority of RNA polymerase II (RNAPII) promoters in mouse embryonic stem cells (mESCs), and this activity correlates with CpG islands. Here we report the characterization of upstream antisense transcription in regions encoding transcription start site associated RNAs (TSSa-RNAs) at four divergent CpG island promoters: Isg20l1, Tcea1, Txn1, and Sf3b1. We find that upstream antisense RNAs (uaRNAs) have distinct capped 5' termini and heterogeneous nonpolyadenylated 3' ends. uaRNAs are short-lived with average half-lives of 18 minutes and are present at 1-4 copies per cell, approximately one RNA per DNA template. Exosome depletion stabilizes uaRNAs. These uaRNAs are probably initiation products because their capped termini correlate with peaks of paused RNAPII. The pausing factors NELF and DSIF are associated with these antisense polymerases and their sense partners. Knockdown of either NELF or DSIF results in an increase in the levels of uaRNAs. Consistent with P-TEFb controlling release from pausing, treatment with its inhibitor, flavopiridol, decreases uaRNA and nascent mRNA transcripts with similar kinetics. Finally, Isg20l1 induction reveals equivalent increases in transcriptional activity in sense and antisense directions. Together these data show divergent polymerases are regulated after P-TEFb recruitment with uaRNA levels controlled by the exosome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Capped antisense RNA from divergent transcription initiate upstream of antisense TSSa-RNAs and display 3′ heterogeneity. UCSC Genome browser view showing the location of detected 5′ and 3′ ends using rapid amplification of cDNA ends (RACE) at four selected CpG island divergent promoter genes: Isg20l1, Tcea1, Txn1, and Sf3b1. 5′-RACE analysis was performed on upstream regions containing more than one overlapping antisense TSSa-RNA (5). Promoter regions are shown with the TSSs marked (arrows pointing in the direction of transcription). Arrows depicting antisense transcription are pointing to the left while sense TSSs are marked with arrows pointing to the right. Sense TSSs (right arrows) were labeled according to UCSC genome browser’s known genes from UniProt, RefSeq, and GenBank. These were independently confirmed by 5′-RACE for Isg20l1, Tcea1, and Txn1. 3′-RACE analysis yielded various uaRNA transcripts (green) for each divergent promoter. These range in length from approximately 50 to 1,100 nts. ChIP-seq binding profiles of RNAPII, H3K4me3, H3K79me2, TATA-binding protein (TBP), TSSa-RNA reads (antisense = blue; sense = red), and CpG island regions (green) are shown. ChIP-Seq data was obtained from the following published reports: RNAPII-8W16 (6), H3K4me3 and H3K79me2 (23), and TBP (22). We note the absense of a sense RNAPII ChIP-seq peak at the annotated TSS of Tcea1, likely explained in part to difficulties in mapping reads to this region since it contains high similarity (99%) with a location on chromosome 15. Scale bars are displayed at the top of each promoter region.
Fig. 2.
Fig. 2.
uaRNA transcripts are substrates for the exosome. (A) Relative levels of uaRNA (gray amplicon in Fig. S2A, dark gray) and spliced mRNA (exon1-2 probe, light gray) transcripts in mESCs infected with virus containing an shRNA targeting Exosc5 (shExosc5) and assayed by RT-qPCR (probes shown in Table S3). Transcript levels were normalized to virus-infected cells without shRNA, empty vector (pLKO.1), and normalized to β-actin levels. Values represent four biological replicates and error represents the respective SEM. Asterisks represent significance of p < 0.05 in two-sided t test. (B) 3′-RACE followed by Southern blot analysis of control and shExosc5 treated mESC RNA. The Southern blots were probed with probe 1 shown in Fig. S6A. Minus RT lanes refer to 3′-RACE experiments with no reverse transcriptase added in the RT step of the procedure. Migration of 100bp molecular weight ladder (NEB) is marked on the left.
Fig. 3.
Fig. 3.
Pausing factors at the antisense RNAPII complex regulate uaRNA levels. (A) UCSC Genome browser views of the four divergent promoter regions displaying ChIP-seq binding profiles (13) of RNAPII-Ser5P, Supt5h, NELF-A, and the full length antisense RNA transcripts. Each region diagrammed spans 2 kb and scale bars represent 500 bp. (B) Relative gene expression of Nelf-A, Nelf-E, and Supt4h in control and shRNA knockdown mESC after 48 h of selection measured by Taqman RT-qPCR assay. Values represent six biological replicates and error represents the respective SEM. (C) Transcript changes in shNELF-A, shNELF-E, and shSupt4h mESC lines as determined by RT-qPCR. uaRNA and spliced mRNA levels are represented by blue and red bars, respectively (probes shown in Table S3). Values represent six biological replicates and error shows the respective SEM. Asterisks represent significance of p < 0.05 in two-tailed t test.
Fig. 4.
Fig. 4.
uaRNAs are P-TEFb-dependent transcripts and have short half-lives. (A) Relative levels of spliced mRNA (red), nascent RNA (exon1-intron1 probe, green), and uaRNA (blue) transcripts, as measured by RT-qPCR, after a 1 μM flavopiridol treatment for 1 h. (B) uaRNA transcript levels assayed by RT-qPCR from amplicon shown in Fig. S2A over a 1 h time course with 1 μM flavopiridol. (C) uaRNA (left panel) and nascent sense RNA (right panel) levels assayed by RT-qPCR over an hour 1 μM flavopiridol treatment followed by a phosphate buffered saline (PBS) wash off of flavopiridol at the indicated times. Isg20l1, Tcea1, Txn1, and Sf3b1 RNA transcripts are shown in blue, red, green, and purple, respectively. All values are relative to mock (DMSO) treated cells and normalized to β-Actin. Values represent two biological replicates with the error representing the respective SEMs. All probe sequences are shown in Table S3.
Fig. 5.
Fig. 5.
Divergent sense and antisense transcription induced with similar kinetics. mESCs were treated with 1 μM doxorubicin for 1.5, 4, and 6 h after which total RNA was collected. RT-qPCR analysis to determine the relative fold change of nascent, spliced mRNA, and uaRNA transcription followed. Changes in transcript levels for two genes, Isg20l1 and Txn1, green and blue bars, respectively, are shown. Values represent biological triplicates and error of the respective SEM. All probes are shown in Table S3.
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