Transient transcriptome sequencing (TT-Seq) and 5'P-Seq of ATP-analog sensitive Kin28 budding yeast

Inhibition of Kin28/CDK7, the kinase subunit of TFIIH, leads to defects in transcription of protein-coding genes. Despite a severe reduction in nascent RNA synthesis, the majority of mRNAs retain their steady-state level upon inhibition. In this study, we examined the determinants of mRNA stability in cells experiencing transcriptional crisis via irreversible chemical inhibition of Kin28. We discovered that the inhibited Kin28 transcriptome resembles the transcriptome of cells treated with an inhibitor of protein synthesis. Indeed, inhibition of Kin28 induces a coordinated decrease in translation and an increase in P-body formation. Unexpectedly, integrated stress response effectors do not trigger the observed proteostasis and ribostasis. Rather, mRNAs that are buffered from degradation display a preference for Ski2 over Nab2 and are differentially sensitive to the 5′-exonuclease Xrn1. These findings reveal that a nuclear kinase, well-known for its role in early stages of RNA synthesis, orchestrates multiple molecular processes in different cellular compartments.作为转录因子IIH（Transcription Factor IIH，TFIIH）的激酶亚基，Kin28/CDK7受到抑制后会引发蛋白编码基因的转录缺陷。尽管新生RNA的合成量大幅降低，但多数mRNA的稳态水平在抑制处理后仍得以维持。本研究通过不可逆化学手段抑制Kin28，探究了细胞遭遇转录危机时mRNA稳定性的决定因素。我们发现，Kin28受抑制后的转录组与经蛋白质合成抑制剂处理的细胞转录组高度相似。实际上，Kin28抑制会协同诱导翻译水平下降，并促进P小体（P-body）的形成。出乎意料的是，整合应激反应效应因子并未触发所观测到的蛋白质稳态与核糖体稳态变化。与之相对，免受降解的mRNA更偏好结合Ski2而非Nab2，且对5'外切核酸酶Xrn1的敏感性存在差异。上述研究结果揭示，这一因在RNA合成早期阶段发挥功能而广为人知的核激酶，能够协调不同细胞区域内的多种分子过程。