SUMMARY Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse biological processes. Here we describe the initial functional analysis of a poorly characterized human lncRNA (LINC00657) that is induced after DNA damage, which we termed Noncoding RNA Activated by DNA Damage or NORAD. NORAD is highly conserved and abundant, with expression levels of approximately 500–1,000 copies per cell. Remarkably, inactivation of NORAD triggers dramatic aneuploidy in previously karyotypically-stable cell lines. NORAD maintains genomic stability by sequestering PUMILIO proteins, which repress the stability and translation of messenger RNAs to which they bind. In the absence of NORAD, PUMILIO proteins drive chromosomal instability by hyperactively repressing mitotic, DNA repair, and DNA replication factors. These findings introduce a mechanism that regulates the activity of a deeply conserved and highly dosage-sensitive family of RNA binding proteins and reveal unanticipated roles for a lncRNA and PUMILIO proteins in the maintenance of genomic stability.

The appropriate timing of events that lead to chromosome segregation during mitosis and cytokinesis is essential to prevent aneuploidy, and defects in these processes can contribute to tumorigenesis. Key mitotic regulators are controlled through ubiquitylation and proteasome-mediated degradation. The Anaphase-Promoting Complex or Cyclosome (APC/C) is an E3 ubiquitin ligase that has a crucial function in the regulation of the mitotic cell cycle, particularly at the onset of anaphase and during mitotic exit. Co-activator proteins, inhibitor proteins, protein kinases and phosphatases interact with the APC/C to temporally and spatially control its activity and thus ensure accurate timing of mitotic events.

SUMMARY Balanced chromosome segregation in mitosis requires synchronous chromatid separation at anaphase and the precise coordination of anaphase with cytokinesis and mitotic exit. The mitotic spindle checkpoint monitors proper attachment and/or tension induced by microtubule binding to sister kinetochores. Within each cell, once all chromosomes achieve bipolar attachment to the spindle poles and align at the metaphase plate, the spindle checkpoint is silenced triggering anaphase onset, cytokinesis, and mitotic exit. We used a bioinformatics approach to identify a candidate protein, C13orf3/Ska3, predicted to function in mitosis. Cells in which Ska3 expression was reduced by RNAi achieved metaphase alignment but were unable to silence the spindle checkpoint and enter normal anaphase. After hours of metaphase arrest, chromatids separated but retained robust attachment to spindle microtubules. These cells remained checkpoint arrested with strong accumulation of the checkpoint protein Bub1 at kinetochores. During normal mitosis Ska3 protein accumulated on kinetochores in prometaphase after nuclear envelope breakdown. This kinetochore localization of Ska3 was dependent on Shugoshin (Sgo1), the “guardian spirit” of chromatid cohesion. In contrast, Sgo1, which accumulates at the centromeres in early prophase, was not dependent on Ska3. Although Ska3 is required for maintenance of sister chromatid cohesion and is dependent upon Sgo1, cells with reduced Sgo1 show a stronger premature chromatid separation phenotype than those with reduced Ska3. We hypothesize that Ska3 functions as a component of a network that coordinates checkpoint signaling from the microtubule binding sites within a kinetochore by laterally linking the individual binding sites. We suggest that this network plays a major role in silencing the spindle checkpoint when chromosomes are aligned at metaphase to allow timely anaphase onset and mitotic exit.

Summary Background Chromosome instability is thought to be a major contributor to cancer malignancy and birth defects. For balanced chromosome segregation in mitosis, kinetochores on sister chromatids bind and pull on microtubules emanating from opposite spindle poles. This tension contributes to the correction of improper kinetochore attachments and is opposed by the cohesin complex that holds the sister chromatids together. Normally, within minutes of alignment at the metaphase plate, chromatid cohesion is released, allowing each cohort of chromatids to move synchronously to opposite poles in anaphase, an event closely coordinated with mitotic exit. Results Here we show that during experimentally induced metaphase delay spindle pulling forces can cause asynchronous chromatid separation, a phenomenon we term “cohesion fatigue.” Cohesion fatigue is not blocked by inhibition of Plk1, a kinase essential for the “prophase pathway” of cohesin release from chromosomes or by depletion of separase, the protease that normally drives chromatid separation at anaphase. Cohesion fatigue is inhibited by drug-induced depolymerization of mitotic spindle microtubules and by experimentally increasing the levels of cohesin on mitotic chromosomes. In cells undergoing cohesion fatigue, cohesin proteins remain associated with the separated chromatids. Conclusion In cells arrested at metaphase, pulling forces originating from kinetochore-microtubule interactions can, with time, rupture normal sister chromatid cohesion. This cohesion fatigue, resulting in unscheduled chromatid separation in cells delayed at metaphase, constitutes a previously overlooked source for chromosome instability in mitosis and meiosis.

Activation of Cdk1 is rapid and switch-like due to positive feedback mechanisms. When Cdk1 is fully on, cells are capable of M-to-G1 transition. Inhibition of positive feedback prevents rapid Cdk1 activation and induces a mitotic “collapse” phenotype characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis.