The Integrator Complex Attenuates Promoter-Proximal Transcription at Protein-Coding Genes

Abstract

The transition of RNA polymerase II (Pol II) from initiation to productive elongation is a central, regulated step in metazoan gene expression. At many genes, Pol II pauses stably in early elongation, remaining engaged with the 25-to 60-nt-long nascent RNA for many minutes while awaiting signals for release into the gene body. However, 15%-20% of genes display highly unstable promoter Pol II, suggesting that paused polymerase might dissociate from template DNA at these promoters and release a short, non-productive mRNA. Here, we report that paused Pol II can be actively destabilized by the Integrator complex. Specifically, we present evidence that Integrator utilizes its RNA endonuclease activity to cleave nascent RNA and drive termination of paused Pol II. These findings uncover a previously unappreciated mechanism of metazoan gene repression, akin to bacterial transcription attenuation, wherein promoter-proximal Pol II is prevented from entering productive elongation through factor-regulated termination.

INTS13Mutations Causing a Developmental Ciliopathy Disrupt Integrator Complex Assembly

“…This finding is consistent with previous studies showing that depletion of multiple Integrator subunits can also lead to disrupted ciliogenesis 41 , and that mutations within INTS1 or INTS8 also create ciliopathy-related symptoms 42,65 . Herein, we discovered that depletion of INTS13 causes a ~2-to 10-fold upregulation in a broad collection of ciliary genes in RPE cells, which is consistent with recent work demonstrating that depletion of Integrator subunits in Drosophila cells primarily causes an increase in transcription in a subset of genes 37,38 . We posit that an increase in transcription of a broad collection of ciliary genes is sufficient to disrupt ciliogenesis by potentially overproducing specific components.…”

“…Denatured protein samples were then resolved on a 10% polyacrylamide gel and transferred to a PVDF membrane (Thermo Scientific, 88518). Blots were probed as previously described 37 . Samples from nuclear extract immunoprecipitation are described in the anti-FLAG affinity purification.…”

“…The Integrator CM, through the activity of INTS11, has been shown to be essential to cleave the 3' ends of a variety of noncoding RNA and nascent mRNA of protein-coding genes with paused RNAPII. This catalytic activity thus serves as a key functional component of Integrator-mediated transcriptional repression and for the biogenesis of noncoding RNA 34,35,36,37,38 .…”

Hyperosmotic stress induces downstream-of-gene transcription and alters the RNA Polymerase II interactome despite widespread transcriptional repression

“…Depletion of Integrator subunits has been shown to alter the transcriptional levels of certain genes (Gardini et al 2014;Elrod et al 2019;Tatomer et al 2019). Consistently, we found that depletion of functional Int11 in HEK-293T cells differentially affects more than a thousand genes ( Figure S6F).…”

Transcript isoform sequencing reveals widespread promoter-proximal transcriptional termination in Arabidopsis

“…As there is known RNAPII accumulation at PASs at the ends of genes, it is possible that the accumulation of RNAPII at the pausing sites is a result of the same process, but coupled to promoter-proximal polyadenylation. Indeed, short transcripts at genes with unstable promoter-promoter proximal RNAPII pausing are reported in Drosophila 29 that rely on transcriptional termination by the Integrator complex 30,31 , that also functions in 3′-end processing of relatively short snRNA 32 . The prevalence of promoterproximal transcriptional termination in additional organisms and potential roles of this phenomenon in gene expression remain an active research area.…”