The regulation of premature termination at divergent promoters

Abstract

Transcription is one of the most fundamental processes in cells, governing the conversion of genetic information to RNA. Numerous regulatory mechanisms function to ensure that desired transcripts are being expressed. Promoters transcribe divergently, producing low-abundant upstream antisense RNAs (uaRNAs) in addition to a stable downstream RNAs. Thus, a central question is what mechanisms are sense RNAs more stable compared to most transcription events. It is proposed that an asymmetric distribution of Ul snRNP binding sites and polyadenylation site (PAS) motifs known as the UI-PAS axis regulates early termination of RNA Polymerase II. Here, we generated a conditional knockout of the essential RNA exosome subunit, Exosc3, in mouse embryonic stem cells. Removal of Exosc3 resulted in stabilization of polyadenylated uaRNAs, enhancer RNAs and long noncoding RNAs. In addition, promoter proximal pausing increased modestly upon Exosc3 removal.

Interestingly, a large class of polyadenylated short transcripts in the sense direction terminate within the first intron, similar to premature termination observed upon Ul inhibition. Further investigation of these prematurely termination sites revealed they are found at the edges of stable nucleosome free regions demarcated by CpG islands and are suppressed by U1 snRNP. Interestingly, promoter-proximal Pol II pausing consists of two processes: TSS-proximal and +1 stable nucleosome pausing. Genes associated with premature termination have increased +1 stable nucleosome pausing, association of chromatin remodelers and are more sensitive to inhibition by flavopiridol or a Myc inhibitor. Additionally, the nuclear poly(A) binding protein, Pabpnl, promotes degradation of polyadenylated uaRNAs. Most Pabpnl sensitive uaRNAs are also Exosc3 substrates, and sensitivity to Pabpnl inhibition inversely correlates with the proximity of the termination site to the TSS.

Interestingly, at uaRNAs and sense RNAs, Pabpnl -sensitive PAS termination events also occur near the first stable nucleosome, similar to Exosc3-sensitive PAS termination events, suggesting that Pabpnl collaborates with Exosc3 to regulate stability of polyadenylated transcripts. Hence, this supports a model whereby Ul snRNP, +1 stable nucleosomes and the degradation machinery converge to create a transcription elongation checkpoint downstream of promoter-proximal pausing.