Kitagawa R: Identification and Characterization of Novel Components of the Spindle Assembly Checkpoint



To identify genes required for the activation of the SAC pathway, we performed a genome-wide RNAi screen for synthetic genetic interactors with CeMAD3, a C. elegans homolog of the conserved SAC component MAD3. CeMAD3 is nonessential in C. elegans. Our primary screen identified 131 synthetic-lethal genes in the CeMAD3-deletion strain, including 12 genes whose functions in chromosome transmission remain unknown. Among those synthetic-lethal genes, we identified one novel gene, tentatively named tsl-1 (Mad3(three) synthetic lethal-1), which is required for spindle damage–induced mitotic delay in embryonic cells. We generated a transgenic strain expressing GFP::TSL-1 and found that TSL-1 localizes at the kinetochore of mitotic chromosomes in embryonic cells. Furthermore, functional analysis using RNAi revealed that TSL-1 is required for proper alignment of mitotic chromosomes during metaphase and for activation of the SAC. Finally, we demonstrated that TSL-1 physically associates with CeMAD1 in vivo, a finding that suggests that TSL-1 recruits CeMAD1 to unattached kinetochores. We will further investigate the role of this gene product in SAC-regulated chromosome segregation and elucidate its molecular function in the SAC-signaling pathway.


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