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Aurora B-Dependent Ndc80 Degradation Regulates bioRxiv preprint doi: https://doi.org/10.1101/836668; this version posted November 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Aurora B-dependent Ndc80 Degradation Regulates Kinetochore Composition in 2 Meiosis 3 4 5 Running title: Aurora B Regulates Ndc80 Proteolysis in Meiosis 6 7 Keywords: meiosis, kinetochore, Aurora B, Ndc80, chromosome, proteolysis, APC 8 9 10 Jingxun Chen1, Andrew Liao1, Emily N Powers1, Hanna Liao1, Lori A Kohlstaedt2, Rena 11 Evans3, Ryan M Holly1, Jenny Kim Kim4, Marko Jovanovic4 and Elçin Ünal1, § 12 13 1 Department of Molecular and Cell Biology, University of California, Berkeley, CA 14 94720, United States 15 2 UC Berkeley QB3 Proteomics Facility, University of California, Berkeley, CA 94720, 16 United States 17 3 Fred Hutchinson Cancer Research Center, Seattle, WA 98109, United States 18 4 Department of Biology, Columbia University, New York City, NY 10027, United States 19 20 § Correspondence: [email protected] 21 22 23 24 25 26 1 bioRxiv preprint doi: https://doi.org/10.1101/836668; this version posted November 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 27 ABSTRACT 28 29 The kinetochore complex is a conserved machinery that connects chromosomes to 30 spindle microtubules. During meiosis, the kinetochore is restructured to accommodate a 31 specialized chromosome segregation pattern. In budding yeast, meiotic kinetochore 32 remodeling is mediated by the temporal changes in the abundance of a single subunit 33 called Ndc80. We have previously described the regulatory events that control the 34 timely synthesis of Ndc80. Here, we report that Ndc80 turnover is also tightly regulated 35 in meiosis: Ndc80 degradation is active in meiotic prophase, but not in metaphase I. 36 Ndc80 degradation depends on the ubiquitin ligase APCAma1 and is mediated by the 37 proteasome. Importantly, Aurora B-dependent Ndc80 phosphorylation, a mark that has 38 been previously implicated in correcting erroneous microtubule–kinetochore 39 attachments, is essential for Ndc80 degradation in a microtubule-independent manner. 40 The N-terminus of Ndc80, including a 27-residue sequence and Aurora B 41 phosphorylation sites, is both necessary and sufficient for kinetochore protein 42 degradation. Finally, defects in Ndc80 turnover predispose meiotic cells to chromosome 43 mis-segregation. Our study elucidates the mechanism by which meiotic cells modulate 44 their kinetochore composition through regulated Ndc80 degradation, and demonstrates 45 that Aurora B-dependent regulation of kinetochores extends beyond altering 46 microtubule attachments. 47 48 49 2 bioRxiv preprint doi: https://doi.org/10.1101/836668; this version posted November 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 50 INTRODUCTION 51 52 Reproduction is a fundamental feature of life and depends on the accurate segregation 53 of chromosomes from one generation to the next. In eukaryotes, a conserved protein 54 complex known as the kinetochore mediates chromosome segregation. Research over 55 the past three decades has identified at least 40 different proteins that constitute the 56 core of this essential machinery (reviewed extensively in Biggins 2013). While the 57 function of individual kinetochore components has been well established, much less is 58 understood about how the levels of specific subunits are regulated under varying 59 cellular states and how these changes affect kinetochore function. 60 The kinetochore is composed of two distinct parts: inner and outer kinetochore. The 61 inner kinetochore subunits associate with the chromosome at the centromere, while the 62 outer kinetochore components interact with spindle microtubules. It has been shown 63 that changes in either part can have a profound impact on kinetochore activity and 64 genome inheritance, with potentially deleterious consequences. For example, 65 overexpression of the centromeric histone CENP-A, a component of the inner 66 kinetochore, in yeast, flies, and human cells causes chromosome mis-segregation and 67 genomic instability (Heun et al. 2006; Au et al. 2008; Shrestha et al. 2017). Additionally, 68 overexpression of the outer kinetochore subunits, such as Hec1 (also known as Ndc80) 69 or SKA1, has been observed in many types of cancers and implicated in tumorigenesis 70 (Chen et al. 1997; Hayama et al. 2006; Li et al. 2014; Shen et al. 2016; Chen et al. 71 2018). 72 Aside from these pathological states, changes to kinetochore composition also occur in 73 physiological contexts. In various organisms, the kinetochore undergoes extensive 74 remodeling during meiotic differentiation (Asakawa et al. 2005; Sun et al. 2011; Miller et 75 al. 2012; Kim et al. 2013; Meyer et al. 2015), which is the developmental program that 76 generates reproductive cells through two consecutive nuclear divisions. Specifically in 77 budding yeast, the Ndc80 complex disassembles in early meiosis and reassembles 78 during the meiotic divisions, thereby restricting kinetochore activity in a temporal fashion 79 (Asakawa et al. 2005; Miller et al. 2012; Meyer et al. 2015; Chen et al. 2017). This 80 dynamic kinetochore behavior is driven by the fluctuating Ndc80 levels, which are barely 81 detectable in meiotic prophase but become highly abundant during the meiotic divisions. 82 Failure to temporally regulate Ndc80 protein levels and kinetochore activity causes 83 defects in meiotic chromosome segregation and gamete inviability (Miller et al. 2012; 84 Chen et al. 2017), highlighting the importance of Ndc80 regulation. 85 One way to regulate Ndc80 protein levels occurs through controlling Ndc80 synthesis. 86 Ndc80 production is relatively high during the meiotic divisions, but is completely shut 87 down in meiotic prophase (Figure 1A) (Chen et al. 2017; Chia et al. 2017). This 88 repression in synthesis requires the expression of a meiosis-specific, 5’-extended 89 mRNA expressed from an alternate NDC80 promoter. This transcript, called LUTI for 90 Long Undecoded Transcript Isoform, is induced by the transcription factor complex 91 Ime1-Ume6 after meiotic entry and cannot be translated into Ndc80 protein. Instead, 92 NDC80LUTI expression acts to interfere with the transcription of the canonical, protein- 3 bioRxiv preprint doi: https://doi.org/10.1101/836668; this version posted November 9, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 93 coding NDC80 mRNA isoform. As a result, in meiotic prophase, a stage when 94 NDC80LUTI is highly expressed, Ndc80 protein synthesis is turned off. After cells exit 95 from meiotic prophase, transcription of the coding NDC80 isoform is induced by another 96 transcription factor called Ndt80, leading to re-synthesis of Ndc80 and kinetochore 97 activation (Chen et al. 2017). Thus, the developmentally coordinated toggling between 98 these two functionally distinct mRNA isoforms controls Ndc80 production in meiosis. 99 The LUTI-based regulation explains how meiotic cells can effectively repress Ndc80 100 protein synthesis. However, since Ndc80 is clearly detected at meiotic entry (Asakawa 101 et al. 2005; Miller et al. 2012; Meyer et al. 2015; Chen et al. 2017), regulated Ndc80 102 synthesis alone cannot fully explain kinetochore inactivation in meiotic prophase. 103 Additional mechanisms must be in place to clear the existing pool of Ndc80 such that 104 the kinetochores can disassemble in a timely manner. Interestingly, the human homolog 105 of Ndc80, Hec1, undergoes degradation in a cell cycle-dependent manner, but the 106 turnover mechanism remains elusive (Ferretti et al. 2010). More generally, little is 107 known about the factors that mediate kinetochore subunit degradation in a 108 developmental context. 109 Here, we describe the mechanism by which Ndc80 degradation is controlled in budding 110 yeast meiosis. We found that the degradation of Ndc80 is temporally regulated. Its 111 proteolysis in meiotic prophase requires Aurora B/Ipl1 kinase-dependent 112 phosphorylation, which has been previously linked to correcting erroneous microtubule– 113 kinetochore attachments (reviewed in Biggins 2013). The N-terminus of Ndc80, 114 including a 27-residue sequence and Ipl1 phosphorylation sites, is both necessary and 115 sufficient for kinetochore protein degradation. In addition to phosphorylation, Ndc80 116 degradation depends on the ubiquitin ligase APCAma1 and proteasome activity. Failure to 117 degrade Ndc80 causes premature kinetochore assembly in meiotic prophase and 118 predisposes cells to meiotic chromosome segregation defects. Our results provide 119 mechanistic insight into how cells can developmentally modulate kinetochore 120 composition through subunit proteolysis and highlight the importance of timely Ndc80 121 degradation in promoting accurate meiotic chromosome
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