Identification of Integrator-PP2A complex (INTAC), an RNA polymerase II phosphatase

Dephosphorylating RNA polymerase II Transcription in metazoans requires coordination of multiple factors to control the progression of polymerases and the integrity of their RNA products. Zheng et al. identified a new dual-enzyme complex called INTAC, which is composed of protein phosphatase 2A (PP2A) core enzyme and the multisubunit RNA endonuclease Integrator. Structural and functional studies show that INTAC functions as a noncanonical PP2A holoenzyme that dephosphorylates the C-terminal domain of RNA polymerase II to attenuate transcription. This study provides a direct connection between PP2A-mediated dephosphorylation and transcriptional regulation, two fundamental cellular processes. Science, this issue p. eabb5872 The Integrator-PP2A complex functions as a dual-enzyme complex in the regulation of RNA polymerase II–mediated transcription. INTRODUCTION RNA cleavage and phosphorylation-dephosphorylation of the RNA polymerase II (Pol II) C-terminal domain (CTD) are two regulatory mechanisms of transcription. The metazoan-specific Integrator is an RNA endonuclease that regulates Pol II–mediated transcription by cleaving a variety of RNAs. Integrator is composed of at least 14 subunits, INTS1 to INTS14, with a molecular weight greater than 1.4 MDa; mechanistic studies are limited to a few structures of isolated domains. Phosphorylation of 52 heptad repeats of Y1S2P3T4S5P6S7 of human Pol II CTD at Ser2, Ser5, and Ser7 involves multistep transcriptional regulation, and a few phosphatases have been identified (Y, Tyr; S, Ser; P, Pro; T, Thr). The serine-threonine protein phosphatase PP2A represents the majority of phosphatase activities in many human tissues and plays a crucial role in a variety of cellular processes. PP2A indirectly regulates gene expression by targeting various signaling pathways. However, it remains elusive whether PP2A directly regulates transcription. RATIONALE In a study of PP2A functions in the nucleus, we found that Integrator associates with the PP2A core enzyme formed by PP2A-A (scaffold subunit) and PP2A-C (catalytic subunit). We reconstituted the human Integrator-PP2A complex and determined the cryo–electron microscopy structure. The chromatin localization of the complex was detected by chromatin immunoprecipitation sequencing. Biochemical and cellular analyses were performed to investigate the binding of Integrator-PP2A to Pol II and dephosphorylation of Pol II CTD. The effect of the phosphatase and endonuclease of INTAC on transcription was tested by structure-guided functional analyses. RESULTS We identified a stable Integrator-containing PP2A-AC complex, which we term INTAC. The 3.5-Å-resolution structure shows that nine human Integrator subunits and PP2A core enzyme assemble into a rigid four-module complex. A cruciform-shaped central scaffold is formed by the backbone (INTS1-INTS2-INTS7) and shoulder (INTS5-INTS8) modules. The phosphatase (INTS6 and PP2A-AC) and endonuclease (INTS4-INTS9-INTS11) modules flank the opposite sides, with the phosphatase (PP2A-C) and endonuclease (INTS11) subunits being apart from each other by as much as ~150 Å. Canonical PP2A holoenzyme consists of the PP2A core enzyme and a single regulatory subunit (PP2A-B). By contrast, INTAC functions as a noncanonical PP2A holoenzyme and Integrator serves as a multicomponent regulatory subunit, by which the PP2A core enzyme is recruited to chromatin-associated Pol II and dephosphorylates Pol II CTD at Ser2, Ser5, and Ser7. The phosphatase of INTAC suppresses transcription of coding genes and noncoding elements and affects transcription initiation, pausing, and elongation. By contrast, the endonuclease of INTAC mainly modulates the pausing-elongation transition and plays a more evident role in regulating the transcription of small nuclear RNAs. Thus, consistent with their architectural separation in the INTAC structure, the RNA endonuclease and phosphatase play distinct roles in the regulation of Pol II–mediated transcription. CONCLUSION We present the identification, structural visualization, and functional characterization of the Integrator-PP2A complex, a noncanonical PP2A holoenzyme. Our study provides a direct connection between PP2A-mediated dephosphorylation and transcriptional regulation, two of the most fundamental cellular processes, and reveals how dual enzymatic activities are structurally and functionally integrated into the INTAC complex. Modular organization and function of INTAC complex. Schematic model of canonical PP2A holoenzymes (top left) and the INTAC complex (top right), which indirectly and directly regulate transcription, respectively. A, B, and C represent the scaffold (PP2A-A), regulatory (PP2A-B), and catalytic subunit (PP2A-C) of a canonical PP2A holoenzyme, respectively, and the red star indicates the phosphatase catalytic cavity; on the right, numbers indicate Integrator subunits. (Bottom) INTAC suppresses transcription through binding Pol II and dephosphorylating Pol II CTD at Ser2, Ser5, and Ser7. The loss of phosphatase activity increases the levels of Pol II phosphorylation and leads to dysregulated transcription of INTAC target genes. pSer2, pSer5, and pSer7 represent the phosphorylated forms of Ser2, Ser5, and Ser7; the numbers indicate Integrator subunits. P-TEFb, the positive transcription elongation factor b. The 14-subunit metazoan-specific Integrator contains an endonuclease that cleaves nascent RNA transcripts. Here, we identified a complex containing Integrator and protein phosphatase 2A core enzyme (PP2A-AC), termed INTAC. The 3.5-angstrom-resolution structure reveals that nine human Integrator subunits and PP2A-AC assemble into a cruciform-shaped central scaffold formed by the backbone and shoulder modules, with the phosphatase and endonuclease modules flanking the opposite sides. As a noncanonical PP2A holoenzyme, the INTAC complex dephosphorylates the carboxy-terminal repeat domain of RNA polymerase II at serine-2, -5, and -7 and thus regulates transcription. Our study extends the function of PP2A to transcriptional regulation and reveals how dual enzymatic activities—RNA cleavage and RNA polymerase II dephosphorylation—are structurally and functionally integrated into the INTAC complex.