Promoter directionality is controlled by U1 snRNP and polyadenylation signals in mouse embryonic stem cells

Abstract

RNA polymerase II (RNAPII) transcription is a tightly regulated process controlling cell type and state. Advancements in our understanding of how transcription is regulated will provide insight into the mechanisms controlling cell identity, cellular differentiation, and its misregulation in disease. It was generally presumed that RNAPII transcribed in a unidirectional manner to produce a coding mRNA. However, RNAPII has recently been found to initiate transcription upstream and antisense from active gene promoters in mammals and yeast. Although RNAPII initiates divergently from these promoters, efficient RNAPII elongation leading to the production of a full-length, stable, abundant RNA molecule is confined to the coding sense direction. These data suggest an unknown mechanism to suppress transcription from the upstream antisense region of divergent promoters. In Chapter 2, we describe an analysis of uaRNA at a candidate set of divergent promoters in mouse embryonic stem cells (mESCs). We reveal that upstream antisense RNAs (uaRNAs) are less than 1 kb in size, 5'-capped, heterogeneous at their 3'-ends, and accumulate to 1-4 copies per cell at the steady state. In addition, uaRNA are transcribed with comparable kinetics as their linked mRNA and undergo RNAPII pausing and pause release via the recruitment and activity of P-TEFb. Furthermore, uaRNA have short half-lives (15-20 minutes), likely due to them being targeted for rapid degradation by the RNA exosome. Altogether, these data indicate that the mechanism regulating promoter directionality at divergent promoters occurs after PTEFb recruitment. In Chapter 3, we describe a genome-wide analysis to map the 3'-ends of polyadenylated RNAs in mESCs and reveal that uaRNAs terminate through a poly (A) site (PAS)-dependent mechanism shortly after being initiated. Interestingly, we find that an asymmetric distribution of encoded U1 snRNP binding sites (U1 sites or 5' splice sites) and PASs surrounding gene transcription start sites (TSSs) enforce promoter directionality by ensuring uaRNAs are prematurely terminated and likely subsequently degraded. Together, these studies highlight the importance of early splicing signals in producing a full-length coding mRNA, but more importantly, our data reveals that the genomic DNA contains the necessary instructions to read the gene in the correct orientation.