Global stabilization of the transcriptome in mitotic cells [PALseq]

In the presence of cell division errors, mammalian cells can pause in mitosis for tens of hours with little to no transcription, while still requiring continued translation for viability. These unique aspects of mitosis require substantial adaptations to the core gene expression programs. During interphase, the homeostatic control of mRNA levels involves a constant balance of transcription and degradation, with a median mRNA half-life of ~2–4 hours. If such short mRNA half-lives persisted in mitosis, cells would be expected to rapidly deplete their transcriptome in the absence of new transcription. Here, we report that the transcriptome is globally stabilized during prolonged mitotic delays. Median mRNA half-lives are increased >4-fold in mitosis compared to interphase, thereby buffering mRNA levels in the absence of new synthesis. Moreover, the poly(A)-tail-length profile of mRNAs changes in mitosis, strongly suggesting a partial mitotic repression of deadenylation. In contrast, the machinery required for siRNA-mediated mRNA degradation remains active. We further show that mitotic mRNA stabilization is dependent on cytoplasmic poly(A)-binding proteins PABPC1&4. Depletion of PABPC1&4 and consequently reduced mRNA stability disrupts the maintenance of mitotic arrest, highlighting the critical physiological role of mitotic transcriptome buffering. Overall design: RNA-seq data from transcription inhibition timecourses in HeLa cells arrested in mitosis, G2, or G1 using double thymidine block followed by STLC, RO3306, or STLC release respectively. One timecourse experiment only contained STLC-arrested cells without additional transcriptional inhibition. Also included are PAL-seq data from the STLC timecourse and the G2-vs-M timecourse in the presence of actinomycin D. RNA-seq data from HCT116 PABPC1&4-AID cells arrested in mitosis, with or without IAA-induced PABPC1&4 depletion (0 h and 4 h post-IAA addition)