Abstract
Ciliogenesis is a fundamental biological process central to human health. Precisely how this process is coordinated with the cell cycle remains an open question. We report that nephrocystin-5 (NPHP5/IQCB1), a positive regulator of ciliogenesis, is a stable and low turnover protein subjected to cycles of ubiquitination and deubiquitination. NPHP5 directly binds to a deubiquitinating enzyme USP9X/FAM and two E3 ubiquitin ligases BBS11/TRIM32 and MARCH7/axotrophin. NPHP5 undergoes K63 ubiquitination in a cell cycle dependent manner and K48/K63 ubiquitination upon USP9X depletion or inhibition. In the G0/G1/S phase, a pool of cytoplasmic USP9X recruited to the centrosome by NPHP5 protects NPHP5 from ubiquitination, thus favouring cilia assembly. In the G2/M phase, USP9X dissociation from the centrosome allows BBS11 to K63 ubiquitinate NPHP5 which triggers protein delocalization and loss of cilia. BBS11 is a resident centrosomal protein, whereas cytoplasmic USP9X sequesters the majority of MARCH7 away from the centrosome during interphase. Depletion or inhibition of USP9X leads to an accumulation of centrosomal MARCH7 which K48 ubiquitinates NPHP5, triggering protein degradation and cilia loss. At the same time, BBS11 K63 ubiquitinates NPHP5. Our data suggest that dynamic ubiquitination and deubiquitination of NPHP5 plays a crucial role in the regulation of ciliogenesis.
Highlights
Primary cilia, microtubule-based protrusions found on the surface of most eukaryotic cells, are derived from centrosomes and possess sensory function such as chemosensation and mechanosensation[1,2]
Defects in cilia formation or ciliogenesis can give rise to a bewildering array of human ciliary diseases collectively known as ciliopathies
Ciliogenesis is controlled in part by nephrocystin-5 (NPHP5/IQCB1), and NPHP5 dysfunction causes ciliopathies in humans, mice and dogs
Summary
Microtubule-based protrusions found on the surface of most eukaryotic cells, are derived from centrosomes and possess sensory function such as chemosensation and mechanosensation[1,2]. Formation of primary cilia is tightly regulated during the cell cycle: they assemble primarily during the G0 phase and undergo complete disassembly prior to entry into mitosis[3]. Defects in cilia formation (ciliogenesis) or function can give rise to a myriad of human genetic disorders collectively known as ciliopathies that are often pleiotropic, exhibiting clinical manifestations such as retinal degeneration, renal failure and neurological disorders[4]. Ciliogenesis is controlled in part by nephrocystin-5 (NPHP5/IQCB1). NPHP5 was originally identified as the causative gene of two human ciliopathies, Senior-Løken syndrome and Leber congenital amaurosis, typified by retinal degeneration with or without renal failure [18,19,20]. We are others have shown that NPHP5 and its interacting partner Cep290 are essential for ciliogenesis[24,25]. Exactly how the stability or activity of this protein is controlled at the molecular level has not been studied
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