otub-3 is a Novel Regulator of Centriole Biogenesis
Date
2024-09-06
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Abstract
Centrioles are organelles in eukaryotic cells with essential functions including organizing the microtubule cytoskeleton and orchestrating mitotic spindle formation. Proper control of centriole number is crucial, as deviations have deleterious consequences. Centriole duplication in Caenorhabditis elegans is initiated in S-phase by the ZYG-1 kinase. Disruption of zyg-1 results in embryonic arrest, as centrioles fail to duplicate. Many negative regulators of centriole biogenesis are suppressors of zyg-1 and can rescue centriole biogenesis and viability in worms carrying a hypomorphic zyg-1 allele. In this thesis, I characterize the role of otub-3, a novel suppressor of zyg-1 identified in a high-throughput genetic screen of zyg-1 in the Tarailo-Graovac lab. I demonstrate that loss of otub-3 rescues viability and centriole duplication in a zyg-1 mutant background without causing centriole amplification in a wildtype background. otub-3 encodes a functional deubiquitinase enzyme and disrupting the catalytic function of OTUB-3 is sufficient to suppress zyg-1. Additionally, loss of otub-3 suppresses spd-2 and zyg-1, but not sas-6, demonstrating genetic relationships with early regulators of centriole biogenesis. A deletion mutant for otub-3 displays normal mitotic timing, suggesting that the interaction between otub-3 and the centriole is independent of the APC/C pathway. OTUB-3 is cytoplasmic and excluded from the nucleus, indicating a potential role in post-translational regulation of centriole regulators. Loss of otub-3 reduces brood size but does not cause embryonic lethality, indicating otub-3 is non-essential and implicated in fertility. Immunoprecipitation followed by mass-spectrometry revealed a physical interaction between OTUB-3 and KIN-3, a known negative regulator of centriole biogenesis. Genetic experiments revealed a genetic interaction between otub-3 and kin-3 as well, positioning otub-3 within a regulatory network functioning to regulate C. elegans centriole biogenesis. This study establishes OTUB-3 as the first deubiquitinase implicated in centriole biogenesis in C. elegans. In addition, this work provides insight into the etiology of human disease. OTUD6B, the human ortholog of otub-3, is linked via an unknown mechanism with primary microcephaly, a genetic disorder commonly caused by centriolar defects. This thesis is the first work to demonstrate a link between an ortholog of OTUD6B and centriole biogenesis, giving a possible mechanism for OTUD6B-linked microcephaly.
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Keywords
Centriole, Forward Genetic Screen, Genetic Modifier, Suppressor, Deubiquitinase, Ubiquitin Biology, Microcephaly
Citation
Nickel, K. (2024). otub-3 is a novel regulator of centriole biogenesis (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.