ARMC5-mediated degradation of RNA polymerase II as a last resort in the promoter-proximal pausing checkpoint [TTchem-Seq]

RNA polymerase II (RNAPII) promoter-proximal pausing is an early step in the transcription cycle, which occurs alongside important events such as mRNA capping. Here, we identify a ubiquitin ligase complex, CUL3-ARMC5 (CRL3ARMC5) which is required for targeting promotor-proximally paused RNAPII for degradation. While a basal level of ARMC5-dependent RNAPII ubiquitylation/degradation occurs even in unperturbed cells, depletion of proteins regulating promoter-proximal pausing results in markedly increased ubiquitylation. We suggest that such RNAPII ubiquitylation/degradation serves as a “last resort” pathway for removing inept polymerases at a promoter-proximal pause checkpoint to avoid their release for unproductive transcript elongation. In agreement with this idea, ARMC5 knockout cells display increased levels of nascent transcription but with pre-mRNAs exhibiting capping defects. Together, our findings support a model in which promoter-proximal pausing functions as a transcription cycle checkpoint and indicate broad role for the CRL3ARMC5 ligase in RNAPII poly-ubiquitylation and degradation. Overall design: We performed TTchem-Seq in WT and ARMC5 KO cells to study how ARMC5 deletion affects genome-wide nascent transcription.

RNA聚合酶II（RNA polymerase II，RNAPII）启动子近端暂停是转录周期中的早期步骤，该过程伴随mRNA加帽等重要事件发生。本研究中，我们鉴定出一种泛素连接酶复合物CUL3-ARMC5（CRL3ARMC5），其可介导启动子近端暂停的RNA聚合酶II的靶向降解。即便在未受干扰的细胞中，也存在基础水平的、依赖ARMC5的RNA聚合酶II泛素化/降解过程；而敲除调控启动子近端暂停的蛋白质后，泛素化水平会显著升高。我们认为，此类RNA聚合酶II的泛素化/降解过程可作为一种"终极补救途径"，在启动子近端暂停检查点清除功能失调的聚合酶，以避免其释放并进行无效转录延伸。与此假说相符的是，ARMC5敲除细胞的新生转录水平有所升高，但前体mRNA存在加帽缺陷。综上，我们的研究结果支持"启动子近端暂停作为转录周期检查点"这一模型，并揭示了CRL3ARMC5连接酶在RNA聚合酶II多泛素化与降解过程中的广泛功能。整体实验设计：我们在野生型（Wild Type，WT）与ARMC5敲除（Knockout，KO）细胞中开展了TTchem-Seq测序，以探究ARMC5缺失对全基因组新生转录的影响。