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.

中文翻译：

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Integrator-PP2A 复合物 (INTAC) 的鉴定，一种 RNA 聚合酶 II 磷酸酶

后生动物中的去磷酸化 RNA 聚合酶 II 转录需要多种因素的协调，以控制聚合酶的进展及其 RNA 产物的完整性。郑等人。鉴定了一种称为 INTAC 的新型双酶复合物，它由蛋白磷酸酶 2A (PP2A) 核心酶和多亚基 RNA 核酸内切酶整合器组成。结构和功能研究表明，INTAC 作为一种非经典的 PP2A 全酶发挥作用，使 RNA 聚合酶 II 的 C 端结构域去磷酸化以减弱转录。这项研究提供了 PP2A 介导的去磷酸化和转录调控之间的直接联系，这两个基本细胞过程。科学，这个问题 p。eabb5872 Integrator-PP2A 复合物作为双酶复合物在 RNA 聚合酶 II 介导的转录调控中发挥作用。介绍 RNA 切割和 RNA 聚合酶 II (Pol II) C 末端结构域 (CTD) 的磷酸化-去磷酸化是两种转录调控机制。后生动物特异性整合子是一种 RNA 核酸内切酶，通过切割各种 RNA 来调节 Pol II 介导的转录。Integrator由至少14个亚基组成，INTS1到INTS14，分子量大于1.4 MDa；机械研究仅限于孤立域的少数结构。人 Pol II CTD 在 Ser2、Ser5 和 Ser7 处的 Y1S2P3T4S5P6S7 的 52 个七肽重复的磷酸化涉及多步转录调控，并且已经鉴定了一些磷酸酶（Y，Tyr；S，Ser；P，Pro；T，Thr）。丝氨酸-苏氨酸蛋白磷酸酶 PP2A 代表了许多人体组织中的大部分磷酸酶活性，并在各种细胞过程中起着至关重要的作用。PP2A 通过靶向各种信号通路间接调节基因表达。然而，PP2A 是否直接调节转录仍然难以捉摸。基本原理 在对细胞核中 PP2A 功能的研究中，我们发现 Integrator 与 PP2A-A（支架亚基）和 PP2A-C（催化亚基）形成的 PP2A 核心酶相关。我们重构了人类 Integrator-PP2A 复合物并确定了低温电子显微镜结构。通过染色质免疫沉淀测序检测复合物的染色质定位。进行生化和细胞分析以研究 Integrator-PP2A 与 Pol II 的结合和 Pol II CTD 的去磷酸化。通过结构引导的功能分析测试了 INTAC 的磷酸酶和核酸内切酶对转录的影响。结果我们确定了一个稳定的包含整合器的 PP2A-AC 复合物，我们称之为 INTAC。3.5 Å 分辨率的结构显示九个人类整合子亚基和 PP2A 核心酶组装成一个刚性的四模块复合体。一个十字形中央支架由主干（INTS1-INTS2-INTS7）和肩部（INTS5-INTS8）模块形成。磷酸酶（INTS6 和 PP2A-AC）和核酸内切酶（INTS4-INTS9-INTS11）模块位于两侧，磷酸酶（PP2A-C）和核酸内切酶（INTS11）亚基彼此相距约 150 Å . 典型的 PP2A 全酶由 PP2A 核心酶和单个调节亚基 (PP2A-B) 组成。相比之下，INTAC 作为一种非经典的 PP2A 全酶和 Integrator 充当多组分调节亚基，通过该亚基，PP2A 核心酶被招募到染色质相关的 Pol II 并在 Ser2、Ser5 和 Ser7 处对 Pol II CTD 进行去磷酸化。INTAC 的磷酸酶抑制编码基因和非编码元件的转录，并影响转录起始、暂停和延伸。相比之下，INTAC的核酸内切酶主要调节暂停-延伸转变，在调节小核RNA的转录中发挥更明显的作用。因此，与它们在 INTAC 结构中的架构分离一致，RNA 核酸内切酶和磷酸酶在 Pol II 介导的转录调控中发挥着不同的作用。结论 我们介绍了 Integrator-PP2A 复合物（一种非规范的 PP2A 全酶）的鉴定、结构可视化和功能表征。我们的研究提供了 PP2A 介导的去磷酸化和转录调控这两个最基本的细胞过程之间的直接联系，并揭示了双重酶活性如何在结构和功能上整合到 INTAC 复合物中。INTAC 综合体的模块化组织和功能。典型的 PP2A 全酶（左上）和 INTAC 复合物（右上）的示意图模型，它们分别间接和直接调节转录。A、B、C分别代表支架（PP2A-A）、监管（PP2A-B）、和一个典型的 PP2A 全酶的催化亚基（PP2A-C），红色星号表示磷酸酶催化腔；在右侧，数字表示积分器子单元。（底部）INTAC 通过结合 Pol II 和在 Ser2、Ser5 和 Ser7 处使 Pol II CTD 去磷酸化来抑制转录。磷酸酶活性的丧失会增加 Pol II 磷酸化的水平，并导致 INTAC 靶基因的转录失调。pSer2、pSer5 和 pSer7 代表 Ser2、Ser5 和 Ser7 的磷酸化形式；数字表示积分器子单元。P-TEFb，正转录延伸因子 b．14 亚基后生动物特异性整合器包含切割新生 RNA 转录物的核酸内切酶。在这里，我们确定了一种复合物，它包含整合器和蛋白磷酸酶 2A 核心酶 (PP2A-AC)，称为 INTAC。3.5 埃分辨率结构显示，九个人类整合子亚基和 PP2A-AC 组装成十字形中央支架，由主干和肩部模块形成，磷酸酶和核酸内切酶模块位于两侧。作为非经典的 PP2A 全酶，INTAC 复合物使 RNA 聚合酶 II 的羧基末端重复结构域在丝氨酸 2、-5 和 -7 处去磷酸化，从而调节转录。我们的研究将 PP2A 的功能扩展到转录调控，并揭示了双重酶活性——RNA 切割和 RNA 聚合酶 II 去磷酸化——如何在结构和功能上整合到 INTAC 复合物中。5 埃分辨率结构显示，九个人类整合子亚基和 PP2A-AC 组装成十字形中央支架，由主干和肩部模块形成，磷酸酶和核酸内切酶模块位于两侧。作为非经典的 PP2A 全酶，INTAC 复合物使 RNA 聚合酶 II 的羧基末端重复结构域在丝氨酸 2、-5 和 -7 处去磷酸化，从而调节转录。我们的研究将 PP2A 的功能扩展到转录调控，并揭示了双重酶活性——RNA 切割和 RNA 聚合酶 II 去磷酸化——如何在结构上和功能上整合到 INTAC 复合物中。5 埃分辨率结构显示，九个人类整合子亚基和 PP2A-AC 组装成十字形中央支架，由主干和肩部模块形成，磷酸酶和核酸内切酶模块位于两侧。作为非经典的 PP2A 全酶，INTAC 复合物使 RNA 聚合酶 II 的羧基末端重复结构域在丝氨酸 2、-5 和 -7 处去磷酸化，从而调节转录。我们的研究将 PP2A 的功能扩展到转录调控，并揭示了双重酶活性——RNA 切割和 RNA 聚合酶 II 去磷酸化——如何在结构上和功能上整合到 INTAC 复合物中。INTAC 复合物使 RNA 聚合酶 II 的羧基末端重复结构域在丝氨酸 2、-5 和 -7 处去磷酸化，从而调节转录。我们的研究将 PP2A 的功能扩展到转录调控，并揭示了双重酶活性——RNA 切割和 RNA 聚合酶 II 去磷酸化——如何在结构上和功能上整合到 INTAC 复合物中。INTAC 复合物使 RNA 聚合酶 II 的羧基末端重复结构域在丝氨酸 2、-5 和 -7 处去磷酸化，从而调节转录。我们的研究将 PP2A 的功能扩展到转录调控，并揭示了双重酶活性——RNA 切割和 RNA 聚合酶 II 去磷酸化——如何在结构上和功能上整合到 INTAC 复合物中。