ARS2 instructs early transcription termination-coupled RNA decay by recruiting ZC3H4 to nascent transcripts [TT-Seq]. ARS2 instructs early transcription termination-coupled RNA decay by recruiting ZC3H4 to nascent transcripts [TT-Seq]

The RNA-binding ARS2 protein is centrally involved in both early RNA polymerase II (RNAPII) transcription termination and transcript decay. Despite its essential nature, the mechanisms by which ARS2 enacts these functions have remained unclear. Here, we show that a conserved basic domain of ARS2 binds a corresponding acidic-rich, short linear motif in the transcription restriction factor ZC3H4. This interaction recruits ZC3H4 to chromatin to elicit Pol II termination, independent of other early termination pathways defined by the cleavage and polyadenylation (CPA) and Integrator (INT) complexes. ZC3H4, in turn, directly connects to the ZCCHC8 component of the nuclear exosome targeting (NEXT) complex, hereby facilitating rapid degradation of the nascent RNA. Hence, ARS2 instructs the coupled transcription termination and degradation of the transcript onto which it is bound. This contrasts with ARS2 function at CPA-instructed termination sites where the protein exclusively partakes in RNA suppression via post-transcriptional decay. Overall design: ARS2, Cleavage and polyadenylation complex subunit CPSF73 (CPSF3), Integrator complex subunit INTS11 and RNA exosome subunit RRP40 (EXOSC3) were knocked down in HeLa cells using siRNA. The corresponding control was the non-tageting EGFP siRNA knockdown. Two biological replicates were generated for transient transcriptome (TT)-seq.

RNA结合蛋白ARS2（RNA-binding ARS2 protein）同时参与早期RNA聚合酶II（RNA polymerase II, RNAPII）转录终止与转录本降解过程，发挥核心调控作用。尽管其功能至关重要，但ARS2介导这些功能的具体分子机制至今仍不明确。本研究发现，ARS2的保守碱性结构域可结合转录限制因子ZC3H4中一段富含酸性氨基酸的短线性基序。该相互作用将ZC3H4招募至染色质，从而介导RNAPII的转录终止，该过程不依赖于由剪切与多聚腺苷酸化复合物（cleavage and polyadenylation, CPA）及Integrator复合物（Integrator, INT）所定义的其他早期终止通路。而ZC3H4可直接与细胞核外泌体靶向复合物（nuclear exosome targeting, NEXT）的组分ZCCHC8结合，进而促进新生RNA的快速降解。由此可见，ARS2可调控其所结合转录本的转录终止与降解这两个偶联过程。这与CPA介导的终止位点处ARS2的功能截然不同：在该位点，ARS2仅通过转录后降解途径参与RNA的抑制调控。实验整体设计：本研究通过siRNA在HeLa细胞中分别敲低ARS2、剪切与多聚腺苷酸化复合物亚基CPSF73（CPSF3）、Integrator复合物亚基INTS11以及RNA外泌体亚基RRP40（EXOSC3）；对应的对照组为靶向EGFP的阴性对照siRNA敲低实验组。针对瞬时转录组测序（transient transcriptome sequencing, TT-seq）设置了两个生物学重复。