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Curr Biol. 2018 Nov 5;28(21):3422-3429.e5. doi: 10.1016/j.cub.2018.10.006. Epub 2018 Oct 25.

Distinct Roles of RZZ and Bub1-KNL1 in Mitotic Checkpoint Signaling and Kinetochore Expansion.

Author information

1
Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.
2
Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
3
Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA; Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
4
Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA. Electronic address: jallepap@mskcc.org.

Abstract

The Mad1-Mad2 heterodimer is the catalytic hub of the spindle assembly checkpoint (SAC), which controls M phase progression through a multi-subunit anaphase inhibitor, the mitotic checkpoint complex (MCC) [1, 2]. During interphase, Mad1-Mad2 generates MCC at nuclear pores [3]. After nuclear envelope breakdown (NEBD), kinetochore-associated Mad1-Mad2 catalyzes MCC assembly until all chromosomes achieve bipolar attachment [1, 2]. Mad1-Mad2 and other factors are also incorporated into the fibrous corona, a phospho-dependent expansion of the outer kinetochore that precedes microtubule attachment [4-6]. The factor(s) involved in targeting Mad1-Mad2 to kinetochores in higher eukaryotes remain controversial [7-12], and the specific phosphorylation event(s) that trigger corona formation remain elusive [5, 13]. We used genome editing to eliminate Bub1, KNL1, and the Rod-Zw10-Zwilch (RZZ) complex in human cells. We show that RZZ's sole role in SAC activation is to tether Mad1-Mad2 to kinetochores. Separately, Mps1 kinase triggers fibrous corona formation by phosphorylating two N-terminal sites on Rod. In contrast, Bub1 and KNL1 activate kinetochore-bound Mad1-Mad2 to produce a "wait anaphase" signal but are not required for corona formation. We also show that clonal lines isolated after BUB1 disruption recover Bub1 expression and SAC function through nonsense-associated alternative splicing (NAS). Our study reveals a fundamental division of labor in the mammalian SAC and highlights a transcriptional response to nonsense mutations that can reduce or eliminate penetrance in genome editing experiments.

KEYWORDS:

aneuploidy; cell division; chromosome segregation; gene editing; spindle assembly checkpoint

PMID:
30415700
PMCID:
PMC6522255
DOI:
10.1016/j.cub.2018.10.006
[Indexed for MEDLINE]
Free PMC Article

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