Center for Eukaryotic Gene Regulation, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Genes Dev. 2019 Jun 1;33(11-12):705-717. doi: 10.1101/gad.322453.118. Epub 2019 Apr 4.
The Ccr4-Not complex regulates essentially every aspect of gene expression, from mRNA synthesis to protein destruction. The Not4 subunit of the complex contains an E3 RING domain and targets proteins for ubiquitin-dependent proteolysis. Ccr4-Not associates with elongating RNA polymerase II (RNAPII), which raises the possibility that it controls the degradation of elongation complex components. Here, we demonstrate that Ccr4-Not controls the ubiquitylation and turnover of Rpb1, the largest subunit of RNAPII, during transcription arrest. Deleting or mutating its RING domain strongly reduced the DNA damage-dependent ubiquitylation and destruction of Rpb1. Surprisingly, in vitro ubiquitylation assays indicate that Ccr4-Not does not directly ubiquitylate Rpb1 but instead promotes Rpb1 ubiquitylation by the HECT domain-containing ligase Rsp5. Genetic analyses suggest that Ccr4-Not acts upstream of , where it acts to initiate the destruction process. Ccr4-Not binds Rsp5 and forms a ternary complex with it and the RNAPII elongation complex. Analysis of mutant Ccr4-Not lacking the RING domain of Not4 suggests that it both recruits Rsp5 and delivers the E2 Ubc4/5 to RNAPII. Our work reveals a previously unknown function of Ccr4-Not and identifies an essential new regulator of RNAPII turnover during genotoxic stress.
Ccr4-Not 复合物基本上调控基因表达的各个方面，从 mRNA 合成到蛋白质降解。该复合物的 Not4 亚基含有一个 E3 RING 结构域，靶向蛋白质进行泛素依赖性蛋白酶体降解。Ccr4-Not 与延伸中的 RNA 聚合酶 II（RNAPII）结合，这提示它可能控制延伸复合物成分的降解。在这里，我们证明 Ccr4-Not 在转录停滞时控制延伸复合物组件的泛素化和周转。删除或突变其 RING 结构域强烈降低了 DNA 损伤依赖性 Rpb1（RNAPII 的最大亚基）的泛素化和破坏。令人惊讶的是，体外泛素化实验表明 Ccr4-Not 并不直接泛素化 Rpb1，而是通过含有 HECT 结构域的连接酶 Rsp5 促进 Rpb1 的泛素化。遗传分析表明 Ccr4-Not 在其上游作用，在那里它启动破坏过程。Ccr4-Not 与 Rsp5 结合，并与它和 RNAPII 延伸复合物形成三元复合物。分析缺乏 Not4 的 RING 结构域的突变 Ccr4-Not 表明它既能募集 Rsp5，又能将 E2 Ubc4/5 递送到 RNAPII。我们的工作揭示了 Ccr4-Not 的一个以前未知的功能，并确定了在遗传毒性应激期间 RNAPII 周转的一个新的必需调节因子。
