The Not4 ubiquitin ligase utilizes its conserved RNA binding domains to regulate global proteostasis and RNA polymerase II-dependent transcription

The conserved Ccr4-Not complex regulates all aspects of the RNA polymerase II (Pol II) gene expression pathway, while it also controls proteasome assembly and function. The Not4 RING domain ubiquitin ligase is a core subunit that also contains an RNA recognition motif (RRM) and C3H1 domain (collectively referred to as the RRM-C) whose function is unknown. Herein, we demonstrate that the Not4 RRM-C contributes to both Not4-dependent regulation of the proteasome and Pol II. Disruption of both Not4 ligase activity and the RRM-C domain simultaneously replicates the proteasome-dependent deubiquitylation and catalytic activity misregulation found in Not4-deficient cells. Transcriptome analysis reveals that the Not4 RRM-C affects a subset of Pol II-dependent genes involved in specific biological functions, including transcription elongation, cyclin-dependent kinase regulated nutrient responses, and ribosomal biogenesis. At these genes, the Not4 RRM-C negatively regulates Pol II binding. While Not4 RRM-C disruption modestly increases between Ccr4-Not and Pol II, a Not4 ligase mutant decreases Ccr4-Not association with Pol II, thus implicating Not4-dependent ubiquitylation in mediating Ccr4-Not interaction with Pol II. Collectively, our results suggests the cellular RNA environment may integrate control of global proteostasis and Pol II transcription through the Not4 ubiquitin ligase.高度保守的Ccr4-Not复合物（Ccr4-Not complex）调控RNA聚合酶II（Pol II）介导的基因表达通路的所有环节，同时还可控制蛋白酶体（proteasome）的组装与功能。Not4环指结构域泛素连接酶（RING domain ubiquitin ligase）是该复合物的核心亚基，同时还携带有RNA识别基序（RRM）与C3H1结构域（C3H1 domain），二者合称为RRM-C结构域，其功能迄今尚未明确。本研究证实，Not4的RRM-C结构域同时参与Not4依赖的蛋白酶体与RNA聚合酶II调控过程。同时破坏Not4连接酶活性与RRM-C结构域，可重现Not4缺陷细胞中出现的、依赖于蛋白酶体的去泛素化与催化活性失调表型。转录组分析结果显示，Not4的RRM-C结构域可影响一类特定的Pol II依赖型基因，这些基因参与多种特定生物学功能，包括转录延伸、细胞周期蛋白依赖性激酶调控的营养响应以及核糖体生物发生。在这些基因位点上，Not4的RRM-C结构域可负向调控Pol II的结合。尽管破坏Not4的RRM-C结构域会轻度增强Ccr4-Not复合物与Pol II之间的相互作用，但Not4连接酶突变体则会降低二者的结合，这表明Not4依赖的泛素化过程介导了Ccr4-Not复合物与Pol II的相互作用。综上，本研究结果表明，细胞内的RNA环境可通过Not4泛素连接酶，协同调控全局蛋白质稳态与Pol II介导的基因转录。