Abstract
Correct segregation of the mitotic chromosomes into daughter cells is a highly regulated process critical to safeguard genome stability. During M phase the spindle assembly checkpoint (SAC) ensures that all kinetochores are correctly attached before its inactivation allows progression into anaphase. Upon SAC inactivation, the anaphase promoting complex/cyclosome (APC/C) E3 ligase ubiquitinates and targets cyclin B and securin for proteasomal degradation. Here, we describe the identification of Ribonucleic Acid Export protein 1 (RAE1), a protein previously shown to be involved in SAC regulation and bipolar spindle formation, as a novel substrate of the deubiquitinating enzyme (DUB) Ubiquitin Specific Protease 11 (USP11). Lentiviral knock-down of USP11 or RAE1 in U2OS cells drastically reduces cell proliferation and increases multipolar spindle formation. We show that USP11 is associated with the mitotic spindle, does not regulate SAC inactivation, but controls ubiquitination of RAE1 at the mitotic spindle, hereby functionally modulating its interaction with Nuclear Mitotic Apparatus protein (NuMA).
Highlights
Ubiquitination, the post-translational modification that results in the addition of minimally one ubiquitin molecule to a protein, regulates a wide range of cellular processes [1]
Large scale Flag-immunoprecipitation (IP) followed by tandem mass spectrometry (MS/MS) analysis identified peptides of Ribonucleic Acid Export protein 1 (RAE1), potassium channel tetramerization domain containing 6 (KCTD6), Protein associated with Myc (PAM, known as Myc binding protein 2, MYCBP2), Citron Rho-Interacting Serine/Threonine Kinase (CIT), Ubiquitin Specific Protease 7 (USP7) and SPRY-domain containing protein 3 (SPRYD3), whilst no corresponding peptides were identified in the negative controls that were analysed in parallel (S1 Fig)
Independent reciprocal IPs using Flag- and hemagglutinin (HA)-tagged versions of the proteins transiently expressed in 293T cells, confirmed the interaction between Ubiquitin Specific Protease 11 (USP11), RAE1, SPRYD3 and KCTD6 (Fig 1A–1E)
Summary
Ubiquitination, the post-translational modification that results in the addition of minimally one ubiquitin molecule to a protein, regulates a wide range of cellular processes [1]. Whilst the addition of ubiquitin chains is a fine-tuned process, the removal by DUBs is well organized. With the goal of identifying novel binding partners and/or substrates of USP11, we performed a proteomics screen using Flag-tagged USP11 as bait and identified RAE1 as a substrate of USP11. Identified as an mRNA export protein [12,13,14], more recent work has shown that RAE1, in complex with Nucleoporin 98 (NUP98), contributes to mitotic checkpoint regulation [15,16,17,18].
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