The Role of Ctk1 Kinase in Termination of Small Non-Coding RNAs

Transcription termination in Saccharomyces cerevisiae can be performed by at least two distinct pathways and is influenced by the phosphorylation status of the carboxy-terminal domain (CTD) of RNA polymerase II (Pol II). Late termination of mRNAs is performed by the CPF/CF complex, the recruitment of which is dependent on CTD-Ser2 phosphorylation (Ser2P). Early termination of shorter cryptic unstable transcripts (CUTs) and small nucleolar/nuclear RNAs (sno/snRNAs) is performed by the Nrd1-Nab3-Sen1 (NNS) complex that binds phosphorylated CTD-Ser5 (Ser5P) via the CTD-interacting domain (CID) of Nrd1p. In this study, mutants of the different termination pathways were compared by genome-wide expression analysis. Surprisingly, the expression changes observed upon loss of the CTD-Ser2 kinase Ctk1p are more similar to those derived from alterations in the Ser5P-dependent NNS pathway, than from loss of CTD-Ser2P binding factors. Tiling array analysis of ctk1Δ cells reveals readthrough at snoRNAs, at many cryptic unstable transcripts (CUTs) and stable uncharacterized transcripts (SUTs), but only at some mRNAs. Despite the suggested predominant role in termination of mRNAs, we observed that a CTK1 deletion or a Pol II CTD mutant lacking all Ser2 positions does not result in a global mRNA termination defect. Rather, termination defects in these strains are widely observed at NNS-dependent genes. These results indicate that Ctk1p and Ser2 CTD phosphorylation have a wide impact in termination of small non-coding RNAs but only affect a subset of mRNA coding genes.

酿酒酵母（Saccharomyces cerevisiae）的转录终止至少存在两种独立途径，其过程受RNA聚合酶II（RNA polymerase II, Pol II）羧基末端结构域（carboxy-terminal domain, CTD）的磷酸化状态影响。mRNA的晚期终止由CPF/CF复合物执行，该复合物的招募依赖于CTD丝氨酸2位点磷酸化（Ser2P）。较短的隐蔽不稳定转录本（cryptic unstable transcripts, CUTs）与小核仁/核内RNA（small nucleolar/nuclear RNAs, sno/snRNAs）的早期终止，则由Nrd1-Nab3-Sen1（NNS）复合物介导，该复合物通过Nrd1p的CTD相互作用结构域（CTD-interacting domain, CID）结合磷酸化的CTD丝氨酸5位点（Ser5P）。本研究通过全基因组表达分析，对不同终止途径的突变体开展了比较研究。令人意外的是，敲除CTD丝氨酸2激酶Ctk1p所引发的转录组表达变化，相较于敲除CTD-Ser2P结合因子所导致的变化，与Ser5P依赖型NNS途径改变引发的表达变化更为相似。对ctk1Δ细胞的平铺阵列（Tiling array）分析结果显示，该突变体在sno/snRNAs、大量隐蔽不稳定转录本（CUTs）与稳定未表征转录本（stable uncharacterized transcripts, SUTs）位点存在转录通读现象，但仅在部分mRNA位点观察到该现象。尽管已有研究提示mRNA终止以CPF/CF途径为主导，但本研究发现，CTK1基因缺失或携带缺失所有丝氨酸2位点的Pol II CTD突变菌株，并不会引发全局性的mRNA终止缺陷。与之相反，此类菌株中的终止缺陷广泛出现在NNS依赖型基因中。上述结果表明，Ctk1p与CTD丝氨酸2位点磷酸化对小型非编码RNA的转录终止具有广泛调控作用，但仅影响部分mRNA编码基因。