Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity.
Xrn2 accelerates termination by RNA polymerase II, which is underpinned by CPSF73 activity.
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DOI：
10.1101/gad.308528.117
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发表时间：
2018-01-15
影响因子：
10.5
通讯作者：
West S
中科院分区：
文献类型：
--
作者：
Eaton JD;Davidson L;Bauer DLV;Natsume T;Kanemaki MT;West S
在这项研究中，Eaton等人使用一种新的基因编辑方法来描述RNA聚合酶II终止的机制。他们产生了可以快速控制5′-to-3′核糖核酸外切酶Xrn 2或poly（A）信号核糖核酸内切酶CPSF 73的人类细胞系，并表明大多数蛋白质编码基因的有效终止涉及CPSF 73介导的RNA切割和Xrn 2对聚合酶相关RNA的共转录降解。终止是每个转录周期中普遍存在的阶段，但尚未完全理解，也是一个争论的主题。我们使用基因编辑作为一种新方法，通过工程化条件性耗尽5′ → 3′核酸外切酶Xrn 2或多聚腺苷酸化信号（PAS）核酸内切酶CPSF 73（切割和多聚腺苷酸化特异性因子73）来解决其机制。快速控制Xrn 2的能力揭示了它在3′侧翼区RNA的共转录降解和转录终止中的明确和普遍作用。这种缺陷的特点是在高分辨率的全基因组使用哺乳动物天然延长转录测序（mNET-seq）。Xrn 2对终止的影响需要事先进行RNA切割，我们通过显示无催化活性的CPSF 73不能恢复缺乏功能性CPSF 73的细胞的终止来提供证据。值得注意的是，Xrn 2在组蛋白或小核RNA（snRNA）基因终止中没有发挥重要作用，即使这两种RNA都经历了3′端切割。总之，大多数蛋白质编码基因的有效终止涉及CPSF 73介导的RNA切割和Xrn 2对聚合酶相关RNA的共转录降解。然而，由于CPSF 73缺失比Xrn 2消除引起更广泛的通读转录，因此它可能在终止中发挥更重要的作用。
In this study, Eaton et al. use a new gene-editing approach to delineate the mechanism by which RNA polymerase II terminates. They generated human cell lines from which the 5′-to-3′ exoribonuclease Xrn2 or the poly(A) signal endoribonuclease CPSF73 can be rapidly controlled and show that efficient termination on most protein-coding genes involves CPSF73-mediated RNA cleavage and cotranscriptional degradation of polymerase-associated RNA by Xrn2. Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5′ → 3′ exonuclease Xrn2 or the polyadenylation signal (PAS) endonuclease CPSF73 (cleavage and polyadenylation specificity factor 73). The ability to rapidly control Xrn2 reveals a clear and general role for it in cotranscriptional degradation of 3′ flanking region RNA and transcriptional termination. This defect is characterized genome-wide at high resolution using mammalian native elongating transcript sequencing (mNET-seq). An Xrn2 effect on termination requires prior RNA cleavage, and we provide evidence for this by showing that catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73. Notably, Xrn2 plays no significant role in either Histone or small nuclear RNA (snRNA) gene termination even though both RNA classes undergo 3′ end cleavage. In sum, efficient termination on most protein-coding genes involves CPSF73-mediated RNA cleavage and cotranscriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive readthrough transcription than Xrn2 elimination, it likely plays a more underpinning role in termination.
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