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）的转录终止过程至少存在两条截然不同的通路，且受RNA聚合酶II（RNA polymerase II, Pol II）羧基末端结构域（carboxy-terminal domain, CTD）的磷酸化状态调控。mRNA的晚期终止由CPF/CF复合物介导，该复合物的招募依赖于CTD丝氨酸2磷酸化（Ser2P）。长度较短的隐秘不稳定转录本（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）复合物执行，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（Ser5P）。本研究通过全基因组表达分析，对不同终止通路的突变菌株进行了比较。令人意外的是，敲除CTD丝氨酸2激酶Ctk1p所引发的表达谱变化，与Ser5P依赖型NNS通路改变所导致的表达变化更为相似，而非敲除CTD-Ser2P结合因子所产生的差异。对ctk1Δ菌株的平铺微阵列分析显示，通读转录现象出现在snoRNA、大量隐秘不稳定转录本（CUTs）以及稳定未表征转录本（stable uncharacterized transcripts, SUTs）的转录区域，但仅在部分mRNA的转录区域被观测到。尽管此前研究提示mRNA终止的主导作用由CPF/CF通路承担，但本研究发现，CTK1基因敲除或缺失Pol II CTD所有丝氨酸2位点的突变菌株，并不会出现全局性的mRNA终止缺陷。相反，这些菌株的终止缺陷广泛存在于NNS通路依赖的基因中。上述结果表明，Ctk1p与CTD丝氨酸2磷酸化对小型非编码RNA的终止过程具有广泛调控作用，但仅对部分mRNA编码基因的终止产生影响。