title: Phylogenomics-guided discovery of a novel conserved cassette of short linear motifs in BubR1 essential for the spindle checkpoint.
abstract: The spindle assembly checkpoint (SAC) maintains genomic integrity by preventing progression of mitotic cell division until all chromosomes are stably attached to spindle microtubules. The SAC critically relies on the paralogues Bub1 and BubR1/Mad3, which integrate kinetochore-spindle attachment status with generation of the anaphase inhibitory complex MCC. We previously reported on the widespread occurrences of independent gene duplications of an ancestral 'MadBub' gene in eukaryotic evolution and the striking parallel subfunctionalization that lead to loss of kinase function in BubR1/Mad3-like paralogues. Here, we present an elaborate subfunctionalization analysis of the Bub1/BubR1 gene family and perform de novo sequence discovery in a comparative phylogenomics framework to trace the distribution of ancestral sequence features to extant paralogues throughout the eukaryotic tree of life. We show that known ancestral sequence features are consistently retained in the same functional paralogue: GLEBS/CMI/CDII/kinase in the Bub1-like and KEN1/KEN2/D-Box in the BubR1/Mad3-like. The recently described ABBA motif can be found in either or both paralogues. We however discovered two additional ABBA motifs that flank KEN2. This cassette of ABBA1-KEN2-ABBA2 forms a strictly conserved module in all ancestral and BubR1/Mad3-like proteins, suggestive of a specific and crucial SAC function. Indeed, deletion of the ABBA motifs in human BUBR1 abrogates the SAC and affects APC/C-Cdc20 interactions. Our detailed comparative genomics analyses thus enabled discovery of a conserved cassette of motifs essential for the SAC and shows how this approach can be used to uncover hitherto unrecognized functional protein features.
authors.affiliation: Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences), Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.|Theoretical Biology and Bioinformatics, Department of Biology, Science Faculty, Utrecht University, 3584 CH, Utrecht, The Netherlands.
authors.forename: Eelco
authors.identifier: 0000-0003-3540-7727
authors.initials: E
authors.lastname: Tromer
authors.affiliation: Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences), Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands.
authors.forename: Debora
authors.initials: D
authors.lastname: Bade
authors.affiliation: Theoretical Biology and Bioinformatics, Department of Biology, Science Faculty, Utrecht University, 3584 CH, Utrecht, The Netherlands b.snel@uu.nl.
authors.forename: Berend
authors.identifier: 0000-0002-5804-8547
authors.initials: B
authors.lastname: Snel
authors.affiliation: Hubrecht Institute-KNAW (Royal Netherlands Academy of Arts and Sciences), Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands g.kops@hubrecht.eu.|Cancer Genomics Netherlands, University Medical Center Utrecht, 3584 CG, Utrecht, The Netherlands.|Center for Molecular Medicine, University Medical Center Utrecht, 3584 CG, Utrecht, The Netherlands.
authors.forename: Geert J P L
authors.identifier: 0000-0003-3555-5295
authors.initials: GJ
authors.lastname: Kops
mesh_terms: D020816:Amino Acid Motifs; D018797:Cell Cycle Proteins; D019143:Evolution, Molecular; D020440:Gene Duplication; D023281:Genomics / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D010802:Phylogeny; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics*; D008941:Spindle Apparatus / Q000235:genetics*
chemical_list: D018797:Cell Cycle Proteins; C584277:BUB1 protein, human; D017346:Protein Serine-Threonine Kinases
keywords: evolutionary genomics; kinetochore; mitosis; short linear motif; spindle checkpoint