GSE125409
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. Overall design: To study the state of transcription and translation in inhibited Kin28 cells, we utilized TT-Seq and 5'P-Seq. Transient transcriptome sequencing (TT-Seq), an optimized RNA-Seq method that combines 4-thiouracil labeling and biotinylation with fragmentation, was utilized because it reduces the 5' bias in reads during capture of nascent transcripts (Schwalb et al., 2016). We also improved the efficiency of recovering labeled transcripts by utilizing a more reactive biotinylation reagent, MTSEA-biotin (Duffy et al., 2015). 5'P-Seq is an RNA-Seq method that selectively captures RNA with a 5'-monophosphate terminus. Ribosome dynamics can be captured by extracting 5'P-Seq signal that displays a 3-nt periodicity in open reading frames (ORFs) (Pelechano et al., 2015).