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The Rae1–Nup98 complex prevents aneuploidy by inhibiting securin degradation

Abstract

Cdc20 and Cdh1 are the activating subunits of the anaphase-promoting complex (APC), an E3 ubiquitin ligase that drives cells into anaphase by inducing degradation of cyclin B and the anaphase inhibitor securin1. To prevent chromosome missegregation, APC activity directed against these mitotic regulators must be inhibited until all chromosomes are properly attached to the mitotic spindle2. Here we show that in mitosis timely destruction of securin by APC is regulated by the nucleocytoplasmic transport factors Rae1 and Nup98. We show that combined Rae1 and Nup98 haploinsufficiency in mice results in premature separation of sister chromatids, severe aneuploidy and untimely degradation of securin. We find that Rae1 and Nup98 form a complex with Cdh1-activated APC (APCCdh1) in early mitosis and specifically inhibit APCCdh1-mediated ubiquitination of securin. Dissociation of Rae1 and Nup98 from APCCdh1 coincides with the release of the mitotic checkpoint protein BubR1 from Cdc20-activated APC (APCCdc20) at the metaphase to anaphase transition. Together, our results suggest that Rae1 and Nup98 are temporal regulators of APCCdh1 that maintain euploidy by preventing unscheduled degradation of securin.

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Figure 1: Combined Rae1/Nup98 haploinsufficiency causes premature ubiquitin-mediated degradation of securin in mitosis.
Figure 2: Rae1 and Nup98 bind to Cdh1-activated APC as a complex in mitosis.
Figure 3: Rae1 and Nup98 inhibit in vitro ubiquitination of securin by Cdh1-activated APC.

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Acknowledgements

We thank B. Fontoura, J. van Ree, D. Baker, M. Dawlaty, D. Katzmann and R. Bram for critically reading the manuscript; M. Thompson, C. Tong, R. Babu, J. Salisbury and R. Pelletier for assistance; M. Kirschner for pCS2–MycCdh1 and pCS2–MycCdh1; J. Chen for antibody against aurora A; S. Melmed and R. Yu for pEGFP–PTTG–N3; J. Pines for pEYFP N1/pds1 KEN–AAA ΔDbox and pEYFP N1/pds1; B. Fontoura for pAltermax UbcH10(C114S); N. Yaseen for pGEX4T3–TEV–Nup98; and G. Mer for help with purifying recombinant proteins. This work was supported by grants from the NIH (to J.v.D.).

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Correspondence to Jan M. van Deursen.

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Supplementary information

Supplementary Figure 1

This figure shows that Rae1+/-/Nup98+/- MEFs express reduced levels of Nup98 and Rae1. (DOC 47 kb)

Supplementary Figure 2

This figure shows examples of chromosome segregation defects seen in Rae1+/-/Nup98+/- MEFs. The figure further lists the percentages of abnormal anaphases of Rae1+/-/Nup98+/-, Rae1+/-, Nup98+/-, and wild-type MEFs. (DOC 754 kb)

Supplementary Figure 3

This figure shows that Rae1+/-/Nup98+/- MEFs are able to activate the spindle assembly checkpoint but unable to sustain it for long periods. The figure further shows that the timing of mitosis is normal in Rae1+/-/Nup98+/- MEFs. (DOC 318 kb)

Supplementary Figure 4

This figure shows immunoblots of mitotic regulators that are normally expressed in Rae1+/-/Nup98+/- MEFs. (DOC 133 kb)

Supplementary Figure 5

This figure shows that securin(δD-box/δKen-box)-YFP is stable in Rae1+/-/Nup98+/- cells. The figure further shows that securin-YFP is unstable in these cells. (DOC 1779 kb)

Supplementary Data

This file describes an experiment that shows that MEFs with low levels of the APC-Cdc20 inhibitor BubR1 degrade cyclin B prematurely, but not securin. (DOC 303 kb)

Supplementary Methods

This file contains a detailed description of all live-cell imaging methods used. (DOC 33 kb)

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Jeganathan, K., Malureanu, L. & van Deursen, J. The Rae1–Nup98 complex prevents aneuploidy by inhibiting securin degradation. Nature 438, 1036–1039 (2005). https://doi.org/10.1038/nature04221

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