Promoter directionality is controlled by U1 snRNP and polyadenylation signals

Asymmetric sequence determinants flanking gene transcription start sites are shown to control directionality of transcription elongation in mammalian cells by regulating promoter-proximal cleavage and polyadenylation. Regulating transcription directionality RNA polymerase II (RNAPII) initiates transcription divergently from most active gene promoters, but an unknown mechanism limits productive elongation to the sense, coding direction. Here, Albert Almada et al . show that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality in mammalian cells. Upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PAS) shortly after their initiation, whereas PAS signals are depleted in the sense direction. Coding genes are enriched in U1 snRNP splice sites that protect against premature cleavage. U1 and PAS sequences therefore define the directionality of transcription elongation and limit pervasive transcription. Transcription of the mammalian genome is pervasive, but productive transcription outside of protein-coding genes is limited by unknown mechanisms 1 . In particular, although RNA polymerase II (RNAPII) initiates divergently from most active gene promoters, productive elongation occurs primarily in the sense-coding direction 2 , 3 , 4 . Here we show in mouse embryonic stem cells that asymmetric sequence determinants flanking gene transcription start sites control promoter directionality by regulating promoter-proximal cleavage and polyadenylation. We find that upstream antisense RNAs are cleaved and polyadenylated at poly(A) sites (PASs) shortly after initiation. De novo motif analysis shows PAS signals and U1 small nuclear ribonucleoprotein (snRNP) recognition sites to be the most depleted and enriched sequences, respectively, in the sense direction relative to the upstream antisense direction. These U1 snRNP sites and PAS sites are progressively gained and lost, respectively, at the 5′ end of coding genes during vertebrate evolution. Functional disruption of U1 snRNP activity results in a dramatic increase in promoter-proximal cleavage events in the sense direction with slight increases in the antisense direction. These data suggest that a U1–PAS axis characterized by low U1 snRNP recognition and a high density of PASs in the upstream antisense region reinforces promoter directionality by promoting early termination in upstream antisense regions, whereas proximal sense PAS signals are suppress