Mitotic progression, arrest, exit or death relies on centromere structural integrity, rather than de novo transcription

Abstract

Recent studies have challenged the prevailing dogma that transcription is repressed during mitosis. Transcription was also proposed to sustain a robust spindle assembly checkpoint (SAC) response. Here, we used live-cell imaging of human cells, RNA-seq and qPCR to investigate the requirement for de novo transcription during mitosis. Under conditions of persistently unattached kinetochores, transcription inhibition with actinomycin D, or treatment with other DNA-intercalating drugs, delocalized the chromosomal passenger complex (CPC) protein Aurora B from centromeres, compromising SAC signaling and cell fate. However, we were unable to detect significant changes in mitotic transcript levels. Moreover, inhibition of transcription independently of DNA intercalation had no effect on Aurora B centromeric localization, SAC response, mitotic progression, exit or death. Mechanistically, we show that DNA intercalating agents reduce the interaction of the CPC with nucleosomes. Thus, mitotic progression, arrest, exit or death is determined by centromere structural integrity, rather than de novo transcription.

Cohesin removal reprograms gene expression upon mitotic entry

“…Retention of elongating RNAPII at centromeres has been observed in metaphase chromosomes of human, fly and frog cells (Blower, 2016;Chan et al, 2012;Molina et al, 2016;Rosic et al, 2014), and several roles for mitotic centromere transcription have been proposed (Blower, 2016;Chan et al, 2012;Liu et al, 2015). However, a recent study has questioned the role of mitotic transcription for accurate chromosome segregation (Novais-Cruz et al, 2018). Our results demonstrate that elongating RNAPII is passively retained at centromeres in mitosis as a byproduct of cohesin retention.…”

“…Importantly, this nascent-RNA signal on mitotic chromosomes in WAPL-AID cells was eliminated by treatment with triptolide, an inhibitor of RNAPII transcription initiation (Titov et al, 2011) (Fig. 2F-G), likely through RNAPII degradation following extended triptolide treatment (Novais-Cruz et al, 2018).…”

The Emerging Role of ncRNAs and RNA-Binding Proteins in Mitotic Apparatus Formation

“…Several studies show that inhibited transcription or splicing in mitosis or degradation of centromeric RNAs results in reduced CENP-C intensity on the kinetochore, anaphase extension, and chromosome segregation errors [22,[73][74][75]. However, a recent study suggested that transcription in mitosis was not required for mitotic progression in human cells [24]. The importance of transcripts in centromeric DNA has been reported for many species, including humans [75], maize [21,76], budding yeast [77], fission yeast [78], and flies [72].…”

“…Since centromeric RNAs are transcribed and accumulated during mitosis [69,80], inhibiting transcription in mitosis affects kinetochore function [69,70]. However, a recent study suggested that transcription in mitosis was not required for mitotic progression [24]. The authors reported that DNA-intercalating drugs (α-amanitin) reduce localization of aurora-B in the centromere without reducing the amount of centromeric RNAs [24].…”

“…The function of ncRNAs transcribed from the centromere region (centromeric RNAs) at the kinetochore has been studied since 2004 and 2007 with maize and human cells, respectively [21,22] Centromeric RNAs are associated with critical kinetochore proteins, recruiting and regulating them at the kinetochore [23]. At present, distinguishing the function of centromeric transcripts and transcription itself in kinetochore formation comes into question [24]. Compared with centrosomes and kinetochores, the function of ncRNAs on the mitotic spindle is poorly understood.…”