The Function And Mechanism Of The Linear Ubiquitin Chain Assembly Complex (LUBAC) In Promoting Ciliogenesis

Primary cilium,presenting on the surface of most vertebrate cells,is a conserved microtubule-based antennae-like sensory organelle that acts on regulations of embryonic development,tissue homeostasis,tumorigenesis,and other important biological processes by receiving and transducing extracellular signals,such as Hedgehog and Wnt signals.When cells enter the quiescent phase（G₀ phase）,primary cilia are assembled outwards from the microtubule of the mother centriole of the centrosome.Primary cilia formation is a precise and well-organised process,including early ciliary vesicles（CV）formation,TTBK2 recruitment of the mother centriole,CP110-CEP97 complex removal from mother centriole,transition zone（TZ）assembly,and elongation of the microtubule axoneme.Defects or dysfunctions of primary cilia lead to a series of severe diseases collectively known as ciliopathies,such as Bardet-Biedl syndrome,Meckel-Gruber syndrome,Joubert syndome,nephronophthisis（NPHP）,polycystic kidney disease,and some neurodegenerative diseases.Currently,it is still difficult for the diagnosis and treatment of ciliopathies,and the pathological mechanisms of these diseases are not completely clear.Therefore,it is important to further understand the molecular mechanism of cilia assembly for the diagnosis and treatment of ciliopathies.Centrosomal protein CP110（Centriolar coiled-coil protein 110）,is the first protein shown to negatively regulate ciliogenesis,localizes at the distal ends of the mother and daughter centriole,and forms a cap-like structure to block the elongation of microtubule axoneme,thereby preventing the inappropriate ciliogenesis in proliferating cells.In the early stage of ciliogenesis,the loss of CP110 from the mother centriole releases the centrosomal role of the mother centriole and promotes the conversion from mother centriole to basal body,which subsequently promotes ciliogenesis.Therefore,the loss of CP110 from the mother centriole is a crucial molecular event at the initial stage of ciliogenesis.Although it has been shown that TTBK2,MARK4,MPP9,and Centrin2are involved in the regulation of CP110 removal from the mother centriole.However,the molecular mechanism that directly regulates the removal of CP110 from the mother centriole remain unclear.The linear ubiquitin chain assembly complex（LUBAC）,consisting of HOIP,HOIL-1L and SHARPIN,is the only known E3 ligase that catalyzes linear ubiquitination（ubiquitin is linked in a head to tail manner）of the substrate proteins.To date,LUBAC mainly functions in the activation of NF-κB signaling pathway by catalyzing the linear ubiquitination of NEMO,RIPK1,TNFR1,and RIPK2,thus promoting inflammatory response.In addition,LUBAC conjugates linear ubiquitin chains to IRF3,ASC,and STAT1 to regulate cell death,inflammasome activation,and antiviral immune response.Despite these advances,in contrast to K48-and K63-linked ubiquitination,linear ubiquitination is less well studied.Therefore,the biological function and physiological significance of linear ubiquitination still need to be further explored.In the early stage of this study,HOIP,the core catalytic subunit of LUBAC,was found to be localized at the centrosome,suggesting that LUBAC may be involved in ciliogenesis.Also,to date,there is no evidence that LUBAC-catalyzed linear ubiquitination functions in cilia assembly.Hence,this study intends to study the role of LUBAC in ciliogenesis at the cellular level and animal level respectively through the technologies of the molecular biology and cell biology,and further explore the molecular mechanism of LUBAC regulating ciliogenesis.First,the overexpression of GFP tagged HOIP protein（GFP-HOIP）in He La cells and RPE-1 cells further confirmed that HOIP is localized to the centrosome.Next,to investigate the role of LUBAC in ciliogenesis,RNAi interference technology was used to deplete LUBAC components in RPE-1 cells,and primary cilia were detected after serum starvation 48 h,the result showed that knockdown of LUBAC strongly inhibited ciliogenesis.Meanwhile,the experiment of respectively supplements with wild-type HOIP and the ligase-inactive mutant C885S（HOIP C885S）proteins in HOIP-depleted cells showed that wild-type HOIP could significantly restore ciliogenesis in HOIP-depleted cell,whereas the ligase-inactive mutant C885S could not.Taken together,these results suggested that LUBAC regulates ciliogenesis and its E3 ligase activity is required for ciliogenesis.It have been reported that the appendage assembly of the mother centriole,ciliary vesicle（CV）formation,TTBK2 recruitment of the mother centriole,and CP110-CEP97complex removal from the mother centriole are crucial for ciliogenesis.Immunofluorescence staining of these processes showed that depletion of LUBAC specifically inhibited the removal of CP110-CEP97 complex from the mother centriole,and knockdown of CP110 in HOIP-depleted cells significantly restored ciliogenesis,suggesting that LUBAC regulates ciliogenesis by promoting the removal of CP110-CEP97 complex from the mother centriole.Furthermore,the linear ubiquitination analysis of CP110 and CEP97 showed that LUBAC specifically catalyzed the linear ubiquitination of CP110,which was further confirmed by in vitro linear ubiquitination analysis of CP110.More importantly,the linear ubiquitination of endogenous CP110 was detected during ciliogenesis.Collectively,these data demonstrated that CP110 is the substrate of LUBAC during ciliogenesis,and also suggested that LUBAC may regulate ciliogenesis by catalyzing the linear ubiquitination of CP110.Expectedly,subsequent studies showed that the abrogation of the linear ubiquitination of CP110 significantly inhibited the removal of CP110 from the mother centriole and ciliogenesis,indicating that LUBAC regulates ciliogenesis through linear ubiquitination of CP110.Furthermore,through mass spectrometry and a series of experiments,PRPF8（Pre-m RNA processing factor 8）was identified as the receptor for the linear ubiquitin chains to drive CP110 dissociation from the mother centriole,thus facilitating ciliogenesis.Finally,zebrafish was used as a research model to study the ciliary functions of LUBAC in vivo.Knockdown of hoip in zebrafish using two different antisense morpholino oligonucleotides（MOs）against hoip led to the defective ciliogenesis in Kupffer’s vesicle（KV）of zebrafish.Furthermore,these hoip-knockdown zebrafish displayed the developmental defects,such as defective left-right asymmetry,curved body,and pericardial edema,demonstrating that LUBAC regulates cilia-related biological processes in vivo.In summary,this study found that LUBAC regulates CP110 removal from the mother centriole by specifically catalyzing the linear ubiquitination of CP110,further studies demonstrated that PRPF8 acts as the receptor for the linear ubiquitin chains of CP110 to drive the removal of CP110 from the mother centriole,thus promoting the ciliogenesis.This study not only reported the crucial role of LUBAC-catalyzed linear ubiquitination in ciliogenesis,also revealed a direct mechanism that regulates CP110removal from the mother centriole,and partially answered the question about how CP110 disappears from the mother centriole in cilia assembly.In addition,the loss of LUBAC in zebrafish causes ciliopathy-related disease phenotypes,such as defective left-right asymmetry,displaying the important physiological function of LUBAC in embryonic development,and also suggesting that the regulation of LUBAC in ciliogenesis may provide new ideas and strategies for the pathological diagnosis and clinical treatment of ciliopathies.