Transcription directionality is licensed by Integrator at active human promoters

Abstract A universal characteristic of eukaryotic transcription is that the promoter recruits RNAPII to produce both precursor mRNAs (pre-mRNAs) and short unstable promoter upstream transcripts (PROMPTs) toward the opposite direction. However, how the transcription machinery senses the correct direction to produce pre-mRNAs is largely unknown. Here, through multiple acute auxin-inducible degradation (AID) systems, we show that rapid depletion of a RNAPII-binding protein complex, Integrator, results in robust PROMPTsaccumulation throughout the genome. Interestingly, the accumulation of PROMPTs is compensated by the reduction of pre-mRNA transcripts in actively transcribed genes. Consistently, Integrator depletion alters the distribution of polymerase between the sense and antisense direction, which is marked by an increased RNAPII-CTD Tyr1 phosphorylation (Tyr1P) level at PROMPT regions and a refrained Ser2 phosphorylation (Ser2P) level at transcription start sites (TSSs). Mechanistically, the endonuclease activity of Integrator is critical to suppress PROMPTs production in a sequence-independent manner. During this step, the endonuclease activity can be inhibited by the U1 signal on nascent antisense transcripts through the recognition of the U1 snRNA-Integrator which relies on the U1-Integrator axis to govern the direction of gene transcription.