靶向RNA聚合酶Ⅱ的蛋白磷酸酶Integrator-PP2A复合体的发现与结构功能研究

The Integrator-PP2A complex functions as an RNA polymerase Ⅱ phosphatase

Integrator复合体是一种特异地存在于多细胞生物体内的多亚基复合体,包含至少14个进化上保守的亚基(INTS1~INTS14)。由于该复合物的组成复杂且样品不易获得,近年来关于Integrator复合体的分子机制的研究相对滞后。我们通过生物化学手段发现,Integrator能够与蛋白磷酸酶PP2A核心酶(PP2A-AC)形成稳定的复合体,并将其命名为INTAC(Integrator-containing PP2A-AC)。该复合体含有16个亚基,相对分子质量近1 500 000。利用冷冻电镜技术,我们首次解析了INTAC的高分辨冷冻电镜结构(整体分辨率3.5 A)。结构显示,核酸酶(endonuclease)和蛋白磷酸酶(phosphatase)这两个催化模块分布在核心的骨架模块两侧。结构、生化和细胞等方面的研究共同揭示了INTAC作为一个双功能酶,除了发挥已知的RNA核酸内切酶的功能,还在转录过程中作用于RNA聚合酶Ⅱ(PolⅡ)中大亚基(Rpb1)的C末端区域(C-terminal domain, CTD),去除其重复七肽中第2、5、7位丝氨酸的磷酸化,并在多个转录过程发挥调控作用。该项研究发现,PP2A这一最重要的磷酸酶可直接调控转录,拓展了转录调控和PP2A这两个重要领域的研究范畴。

The metazoan-specific Integrator, a 14-subunit complex harboring an RNA endonuclease activity, cleaves and processes different classes of RNAs. However, structural and functional studies of Integrator are limited owing to its compositional complexity and sample scarcity. We identified a stable complex composed of Integrator and PP2A core enzyme(termed INTAC). The 3.5 ? cryo-electron microscopy(cryoEM) structure of INTAC reveals a rigid complex organized by the Backbone, Shoulder, Endonuclease, and Phophatase modules. INTAC serves as a noncanonical PP2A holozyme, in which the Integrator portion functions as a regularoty subunit and recruits substrates including RNA polymerase Ⅱ(Pol Ⅱ) whereas the PP2A core enzyme dephosphorylates Pol Ⅱ c-terminal domain(CTD). Disrupting the phosphatase module leads to an aberrant elevation of Pol Ⅱ phosphorylation and thus dysregulation of transcription, an outcome distinct from that of impairing the known RNA endonuclease activity of Integrator. Our study demonstrates how dual enzymatic activities are structurally and functionally integrated into the INTAC complex and provides a direct connection between PP2A-mediated dephosphorylation and transcriptional regulation.