The BUBR1 pseudokinase domain promotes efficient kinetochore PP2A-B56 recruitment to regulate spindle checkpoint silencing and chromosome alignment

ABSTRACTThe balance of phospho-signalling at outer-kinetochores during mitosis is critical for the accurate attachments between kinetochores and the mitotic spindle and timely exit from mitosis. In humans, a major player in determining this balance is the PP2A-B56 phosphatase which is recruited to the Kinase Attachment Regulatory Domain (KARD) of the Spindle Assembly Checkpoint protein Budding Uninhibited by Benzimidazole 1-related 1 (BUBR1) in a phospho-dependent manner. This event unleashes a rapid, switch-like phosphatase relay that reverses phosphorylation at the kinetochore, extinguishing the checkpoint and promoting anaphase entry. Here, we conclusively demonstrate that the pseudokinase domain of human BUBR1 lacks phosphotransfer activity and that it was maintained in vertebrates because it allosterically promotes KARD phosphorylation. Mutation or removal of this domain results in decreased PP2A-B56 recruitment to the outer kinetochore, attenuated checkpoint silencing and errors in chromosome alignment as a result of imbalance in Aurora B activity. We demonstrate that the functions of the BUBR1 pseudokinase and the BUB1 kinase domains are intertwined, providing an explanation for retention of the pseudokinase domain in certain eukaryotes.

Cyclin B1-Cdk1 binding to MAD1 links nuclear pore disassembly to chromosomal stability

How the cell completely reorganises its architecture when it divides is a problem that has fascinated researchers for almost 150 years. We now know that the core regulatory machinery is highly conserved in eukaryotes but how these multiple protein kinases, protein phosphatases, and ubiquitin ligases are coordinated to remodel the cell in a matter of minutes remains a major question. Cyclin B-CDK is the primary kinase that drives mitotic remodelling and here we show that it is targeted to the nuclear pore complex (NPC) by binding an acidic face of the spindle assembly checkpoint protein, MAD1. This localised Cyclin B1-CDK1 activity coordinates NPC disassembly with kinetochore assembly: it is needed for the proper release of MAD1 from the embrace of TPR at the nuclear pore, which enables MAD1 to be recruited to kinetochores before nuclear envelope breakdown, thereby strengthening the spindle assembly checkpoint to maintain genomic stability.

Selective defects in gene expression control genome instability in yeast splicing mutants

RNA processing mutants have been broadly implicated in genome stability, but mechanistic links are often unclear. Two predominant models have emerged: one involving changes in gene expression that perturb other genome maintenance factors and another in which genotoxic DNA:RNA hybrids, called R-loops, impair DNA replication. Here we characterize genome instability phenotypes in yeast splicing factor mutants and find that mitotic defects, and in some cases R-loop accumulation, are causes of genome instability. In both cases, alterations in gene expression, rather than direct cis effects, are likely to contribute to instability. Genome instability in splicing mutants is exacerbated by loss of the spindle-assembly checkpoint protein Mad1. Moreover, removal of the intron from the α-tubulin gene TUB1 restores genome integrity. Thus, differing penetrance and selective effects on the transcriptome can lead to a range of phenotypes in conditional mutants of the spliceosome, including multiple routes to genome instability.

Centromeric CENP-A loading requires accurate mitotic timing, which is linked to checkpoint proteins

A defining feature of centromeres is the presence of the histone H3 variant CENP-A that replaces H3 in a subset of centromeric nucleosomes. In Drosophila cultured cells CENP-A deposition at centromeres takes place during the metaphase stage of the cell cycle and strictly depends on the presence of its specific chaperone CAL1. How CENP-A loading is restricted to mitosis is unknown. We found that overexpression of CAL1 is associated with increased CENP-A levels at centromeres and completely uncouples CENP-A loading from mitosis. Moreover, CENP-A levels inversely correlate with mitosis duration. We found that CAL1 interacts with the spindle assembly checkpoint protein and RZZ complex component Zw10 and thus constitutes the anchor for the recruitment of RZZ. Therefore, CAL1 controls CENP-A incorporation at centromeres both quantitatively and temporally, connecting it to the spindle assembly checkpoint to ensure mitotic fidelity.

The yeast core spliceosome maintains genome integrity through R-loop prevention and α-tubulin expression

To achieve genome stability cells must coordinate the action of various DNA transactions including DNA replication, repair, transcription and chromosome segregation. How transcription and RNA processing enable genome stability is only partly understood. Two predominant models have emerged: one involving changes in gene expression that perturb other genome maintenance factors, and another in which genotoxic DNA:RNA hybrids, called R-loops, impair DNA replication. Here we characterize genome instability phenotypes in a panel yeast splicing factor mutants and find that mitotic defects, and in some cases R-loop accumulation, are causes of genome instability. Genome instability in splicing mutants is exacerbated by loss of the spindle-assembly checkpoint protein Mad1. Moreover, removal of the intron from the α-tubulin gene TUB1 restores genome integrity. Thus, while R-loops contribute in some settings, defects in yeast splicing predominantly lead to genome instability through effects on gene expression.