The Living Systems Institute, University of Exeter, Exeter EX4 4QD, United Kingdom.
Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
Genes Dev. 2018 Jan 15;32(2):127-139. doi: 10.1101/gad.308528.117. Epub 2018 Feb 8.
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.
终止是每个转录周期中普遍存在的阶段，但人们对此了解不完全，也是一个争议的话题。我们使用基因编辑作为一种新方法，通过工程条件性耗尽 5'→3'外切核酸酶 Xrn2 或多聚腺苷酸化信号内切核酸酶 CPSF73（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 Xrn2 的能力清楚地表明了它在转录后 3'侧翼区域 RNA 和转录终止的共转录降解中的普遍作用。使用哺乳动物天然延伸转录物测序 (mNET-seq) 以高分辨率在全基因组范围内对这种缺陷进行了表征。Xrn2 对终止的影响需要先前的 RNA 切割，我们通过证明无活性的 CPSF73 不能将终止恢复到缺乏功能性 CPSF73 的细胞中来提供证据。值得注意的是，Xrn2 对组蛋白或小核 RNA（snRNA）基因的终止没有显著作用，尽管这两种 RNA 类别都经历 3'末端切割。总之，大多数蛋白质编码基因的有效终止涉及 CPSF73 介导的 RNA 切割和 Xrn2 对聚合酶相关 RNA 的共转录降解。然而，由于 CPSF73 缺失引起的通读转录比 Xrn2 消除引起的更多，因此它可能在终止中发挥更基础的作用。
