Defects in a coordination between kinetochore and transition zone cause classical Joubert syndrome

Abstract

Primary cilia are evolutionarily conserved organelles composed of nine outer doublets of microtubule and protruded outside from cell membrane, and their functions are detection of chemical and mechanical signal from extracellular environment. Primary cilia formation are interconnected with numerous cellular and molecular mechanisms such as actin dynamics, cell cycle and cell migration, suggesting that large numbers of regulators work in ciiliogenesis. To identify novel regulators of ciliogenesis, we performed a small-scale siRNA library screen and identified CCDC41 (CEP83), an uncharacterized protein composed of multiple coiled-coil domains. CCDC41 specifically localizes to the distal end of the mother centriole and interacts with the distal appendage component Cep164. Remarkably, knockdown of CCDC41 inhibits the recruitment of IFT20 to the centrosome. Moreover, depletion of either CCDC41 inhibits ciliogenesis at the ciliary vesicle docking step. These study revealed that CCDC41 plays a role as a linker between the mother centriole and other ciliary proteins during the initial stage of ciliogenesis. Recently, biallelic mutations of CCDC41 were identified in seven families. Early-onset nephronophthisis was observed in all affected individuals and learning disability and/or hydrocephalus were displayed in four out of eight individuals. We revealed that whereas wild-type CCDC41 protein showed centrosomal localization and dominant-negative effects on CEP164 localization, CCDC41 p.Gln692del, p.Glu669Aspfs*14 and p.Glu684del variants accumulated into the nucleus and failed to localize at the centrosome. We revealed that this mis-localization of CCDC41 induces that excessive ac-cumulation of CEP164 in centrosome and the excess inhibits the ciliogenesis. Defects in structures and functions of primary cilia cause a range of hereditary disorders called ciliop-athies, such as nephronophthisis, Bardet-Biedl syndrome, Joubert syndrome and Meckel-Gruber syndrome. Because primary cilia play key roles in cerebellar development, mechano-, chemo- and photo-sensation, their defective phenotypes emerge as a variety of developmental and degenerative disorders in diverse organs. In order to identify mutated genes in Korean individuals presumed ciliopathies, we performed a trio-based NGS approaches on affected individuals. Four individuals with ciliopathies, but not detected mutation in known genes associated with ciliopathies were enrolled. As a result, we have identified three genes with a rare variant from separate Joubert syndrome patients. One affected individual carried an X-linked recessive mutation of OFD1 at the splice acceptor site (c.1654-5G>C) with high conservation score. Different biallelic mutations (c.518C>G, c.8113C>T) of gene encoded C5orf42 protein were identified in another individual. A nonsynon-ymous substitution (c.3272G>T) in SHROOM2 gene was identified in the other patient. Among mutated genes, the specific function of C5orf42 remains elusive. Accordingly, we have investigated the function of C5orf42 and the pathologic mechanism of C5orf42 impairment. Here, we revealed that C5orf42 (JBTS 17, OFD6) dynamically localizes to the nuclear pore and the kinetochore in a cell cycle dependent manner. Exog-enously expression of the evolutionarily conserved domain of C5orf42 (Joubert syndrome associated con-served domain, JCD) inhibits the mitotic entry by blocking NEBD and mitotic regulation at the kinetochore. Depletion of C5orf42 displays a prolonged mitosis and defects in cell cycle regulation and chromosome seg-regation, and induces the failure of mitotic arrest by inhibiting the increase of the spindle assembly check-point protein BUBR1 and by inducing defects in a coordination of the mitotic checkpoint proteins such as AurkB and, PLK1. Moreover, NEK6, a regulator of mitotic progression directly interacts with JCD. These re-sults suggest that C5orf42, a ciliopathy protein associated with Joubert syndrome and OFD VI, regulates the mitotic progression by coordinating the spindle assembly checkpoint activation and the assem-bly/disassembly of the nuclear envelope. This incongruity may cause the cilia associated developmental dis-orders, including Joubert syndrome and its related disorders.