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

Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We have 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. The ability to rapidly control Xrn2 reveals a clear and general role for it in co-transcriptional degradation of 3’ flanking region RNA and transcriptional termination. This defect is characterised genome-wide, at high resolution, using 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 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 co-transcriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive read-through transcription than Xrn2 elimination, it likely plays a more underpinning role in termination. Overall design: We performed total CTD mNET-seq and nuclear RNA-seq with auxin-inducible degron(AID) tagged cell lines treated with or without auxin to induce rapid depletion of XRN2. Cell lines ectopically express TIR1 from a sleeping beauty transposon inserted cassette. AID tagged proteins contain the inframe insertion of AID tag, P2A cleavage site, antibiotic resistance marker and SV40 polyA site. Two repair templates containing different antibiotic resistance markers (hygromycin and neomycin) were used to ensure biallelic insertion of the cassette by CRISPR/Cas9.

转录终止是所有转录循环中普遍存在的阶段，但其机制尚未完全明晰，且仍存在学术争议。我们采用基因编辑技术，通过工程化条件性敲除5’-3’外切核酸酶（5’-3’ exonuclease）Xrn2或多聚腺苷酸化信号（polyadenylation signal, PAS）内切核酸酶CPSF73，以探究转录终止的分子机制。可快速调控Xrn2的实验体系，清晰揭示了其在3’侧翼区域RNA的共转录降解及转录终止过程中的普遍功能。我们借助高分辨率全基因组新生延伸转录本测序（native elongating transcript sequencing, mNET-seq）技术，对该缺陷开展了全基因组水平的表征。Xrn2对转录终止的调控作用依赖于预先发生的RNA切割，我们通过实验证实，催化失活的CPSF73无法为缺失功能性CPSF73的细胞恢复转录终止能力，以此为该结论提供了直接证据。值得注意的是，尽管组蛋白基因与小核RNA（snRNA）基因的转录产物均会发生3’端切割，但Xrn2在这两类基因的转录终止过程中均未发挥显著作用。综上，大多数蛋白编码基因的高效转录终止依赖于CPSF73介导的RNA切割，以及Xrn2对聚合酶结合RNA的共转录降解。但相较于敲除Xrn2，缺失CPSF73会引发更为严重的通读转录，因此CPSF73在转录终止过程中可能发挥更为核心的基础性作用。

实验设计：我们对携带生长素诱导型降解（auxin-inducible degron, AID）标签的细胞系开展总CTD新生延伸转录本测序（total CTD mNET-seq）与核RNA测序，分别施加或不施加生长素以快速降解XRN2。该类细胞系通过睡美人转座子（sleeping beauty transposon）插入的外源表达盒异位表达TIR1蛋白。携带AID标签的蛋白，其编码序列中插入了框内的AID标签、P2A自剪切位点、抗生素抗性标记及SV40多聚腺苷酸化位点。我们使用两种携带不同抗生素抗性标记（潮霉素与新霉素）的修复模板，通过CRISPR/Cas9技术实现表达盒的双等位基因插入。