A universal characteristic of eukaryotic transcription is that the promoter recruits RNA polymerase II (RNAPII) to produce both precursor mRNAs (pre-mRNAs) and short unstable promoter upstream transcripts (PROMPTs) toward the opposite direction. However, how the transcription machinery selects the correct direction to produce pre-mRNAs is largely unknown. Here, through multiple acute auxin-inducible degradation systems, we show that rapid depletion of an RNAPII-binding protein complex, Integrator, results in robust PROMPT accumulation 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 directions, which is marked by increased RNAPII-carboxy-terminal domain Tyr1 phosphorylation at PROMPT regions and a reduced Ser2 phosphorylation level at transcription start sites. Mechanistically, the endonuclease activity of Integrator is critical to suppress PROMPT production. Furthermore, our data indicate that the presence of U1 binding sites on nascent transcripts could counteract the cleavage activity of Integrator. In this process, the absence of robust U1 signal at most PROMPTs allows Integrator to suppress the antisense transcription and shift the transcriptional balance in favor of the sense direction.

真核转录的一个普遍特征是启动子招募 RNA 聚合酶 II (RNAPII) 以产生前体 mRNA (pre-mRNA) 和朝相反方向的短不稳定启动子上游转录物 (PROMPT)。然而，转录机制如何选择正确的方向来产生前体 mRNA，目前尚不清楚。在这里，通过多个急性生长素诱导的降解系统，我们表明 RNAPII 结合蛋白复合物 Integrator 的快速消耗会导致整个基因组中大量的 PROMPT 积累。有趣的是，PROMPT 的积累是通过活跃转录基因中前 mRNA 转录物的减少来补偿的。一致地，Integrator 耗竭改变了聚合酶在有义和反义方向之间的分布，其标志是 PROMPT 区域的 RNAPII 羧基末端结构域 Tyr1 磷酸化增加和转录起始位点的 Ser2 磷酸化水平降低。从机制上讲，Integrator 的核酸内切酶活性对于抑制 PROMPT 的产生至关重要。此外，我们的数据表明新生转录本上 U1 结合位点的存在可以抵消 Integrator 的切割活性。在此过程中，大多数 PROMPT 处缺乏强大的 U1 信号，使得 Integrator 能够抑制反义转录并将转录平衡转向有义方向。