Structural basis of INTAC-regulated transcription

Abstract

For the majority of expressed eukaryotic genes, RNA polymerase II (Pol II) forms a paused elongation complex (PEC) and undergoes promoter-proximal pausing downstream of the transcription start site 1–3. The polymerase either proceeds into productive elongation or undergoes promoter-proximal premature transcription termination 4–6. It remains incompletely understood how transcription is regulated at this stage. Here, we determined the structure of PEC bound to INTAC, an Integrator-containing PP2A complex 7, at near-atomic resolution. The structure shows that INTAC partially wraps around PEC through multiple contacts, permitting the memetic nascent RNA to run into substrate-entry tunnel of the endonuclease subunit INTS11 of INTAC for cleavage. Pol II C-terminal domain (CTD) winds over INTAC backbone module through multiple anchors and is suspended above the phosphatase of INTAC for dephosphorylation. Biochemical analysis shows that INTAC-PEC association requires unphosphorylated CTD and could tolerate CTD phosphorylation, suggesting an INTAC-mediated persistent CTD dephosphorylation followed by reinforcement of the INTAC-PEC complex. Our study reveals how INTAC binds PEC and orchestrates RNA cleavage and CTD dephosphorylation, two critical events in generating premature transcription termination.

The PAF1 complex promotes 3′ processing of pervasive transcripts

“…Previous studies of Integrator recruitment focused on its recruitment to snRNA genes by the RNA Pol II CTD and NELF (Egloff et al, 2007(Egloff et al, , 2010Stadelmayer et al, 2014;Yamamoto et al, 2014). These earlier studies were supported by structural work on Integrator-bound PEC comprised of RNA Pol II and pausing factor NELF (Fianu et al, 2021;Zheng et al, 2021), consistent with a model where Integrator is recruited to promoters of snRNAs and shorter, promoter-proximal, non-coding RNAs by PEC. Unlike Integrator depletion, PAF1 depletion did not regulate these shorter RNAs, suggesting that the PAF1C-regulated longer transcripts are regulated differently from the shorter Integrator substrates processed by Integrator-PEC.…”

“…More recently, multiple lines of evidence have demonstrated that Integrator functions in terminating PROMPT and eRNA transcription through cleavage of the nascent RNA products and in attenuating coding gene transcription by cleaving short nascent RNAs (Elrod et al, 2019;Lykke-Andersen et al, 2021;Tatomer et al, 2019). Recruitment of Integrator to short nascent transcripts produced at pause sites and at snRNA genes is through the paused transcription complex (PEC) (Egloff et al, 2007;Fianu et al, 2021;Stadelmayer et al, 2014;Yamamoto et al, 2014;Zheng et al, 2021). However, eR-NAs and PROMPTs can be much longer than these short RNAs.…”

Emerging insights into the function and structure of the Integrator complex

“…The MBL/β-CASP domain of INTS11 is a bona fide nuclease, but remains in an inactive state until its recruitment to paused RNAPII [ 11 , 18 ]. Two recent cryo-EM structures [ 11 , 14 ] revealed that the KOWx-4 domain of SPT5 (DSIF) pushes against the β-CASP lid of INTS11 ( Figure 3b-c ), opening it and allowing a substrate to engage with the active center [ 11 ]. The RNA exiting the active site of RNAPII is partially protected by the KOWx-4 domain of SPT5 and is guided directly into the active site of INTS11.…”

“…Binding of RNAPII CTD to the Integrator is mediated via a composite interface of INTS4/2/7, which forms a cavity that accommodates the RNAPII CTD with sidechain-specific contacts. Locations of additional peptides are reported by Zheng et al with increasing proximity to the active site of PP2A, suggesting a preferred path of the RNAPII CTD within the Integrator complex [ 14 ]. However, no density could be observed close to the active site of PP2AC [ 11 ].…”

“…Although the composition of Integrator was well-established over the years, until recently, there was very little information about its general architecture and molecular structure [ 16 , 17 ]. A recent influx of crystallographic and cryo-EM data provided structural information for almost all known Integrator subunits, including the Integrator cleavage module consisting of INTS4/9/11 [ 18 ], the INTS13/14 heterodimer [ 19 ], the Integrator-PP2A complex [ 20 ], and the Integrator-PP2A bound to RNAPII [ 11 , 14 ]. Together, these structures provide unprecedented mechanistic insights into the spatial arrangement of Integrator’s subunits and their respective functions.…”