Molecular Mechanism Of USP21-mediated Photoreceptor Ciliogenesis

The retina is a transparent membrane that covers the most medial side of the eye and is responsible for converting external optical stimuli into nerve signals for transmission to the brain.The retina is divided into ten layers,among which the photoreceptor cell layer plays a critical role in the process of photoelectric signal transduction.Thus,the retinal structure,especially the development of the photoreceptor cell layer,is very important for the formation of the visual system.Primary cilia are microtubule-based cellular structures that extend from the surface of most mammalian cells and play an important role in detecting external stimuli,signal transduction and cell cycle regulation.The outer segments of photoreceptor cells are usually considered as specialized primary cilia,which are connected to the inner segment through the transition zone.Most components of photoreceptor cells are synthesized in the inner segment and transported along the ciliary axoneme to the outer segment to execute their functions.Therefore,ciliogenesis plays an important role in the development of the photoreceptor cell layer.Ubiquitin specific protease 21(USP21)is a deubiquitinase distributed in the nucleus and cytoplasm.By removing the polyubiquitin chain,USP21 stabilizes the substrate protein and participates in various physiological and pathological activities.In addition to the deubiquitinase domain,USP21 contains a centrosome-binding region and a microtubule binding region,which can directly bind to centrosomes and microtubules.However,it is unclear whether USP21 regulates the formation of primary cilia.The localization and function of USP21 in primary cilia are also mysterious.In addition,it remains to be investigated whether USP21 plays a role in the development of photoreceptor cilia.In this study,transcriptomics analysis was performed at different time points in the development of retina,including the time points right before and after the formation of photoreceptor cell layer.We found that USP21 expression increased significantly along with the retinal development,and get to the peak during the photoreceptor cell development.We constructed USP21 systemic knockout mice and performed electrophysiological and fundus structural examinations.It was found that USP21 deletion resulted in impaired retinal photoreceptor functions,significantly thinner photoreceptor cell layer,and fundus yellow-spotted lesions.Using HE staining and immunofluorescence staining of the retina,we also confirmed that USP21 deletion obstruct the development of the photoreceptor cell layer.The transmission electron microscopy results demonstrated that the ciliary structure of the photoreceptor cells was incomplete.Statistical analysis showed that USP21 deletion significantly reduced the number of cilia of the photoreceptor cells.This part of study shows that USP21 regulates ciliary formation of photoreceptor cells and affects the development of photoreceptor cells and even the retina.Next,we investigated the molecular mechanism of USP21 in regulating primary cilia.We used the retinal tissue treated with USP21 antibody for mass spectrometry experiments,and compared the proteomics expression in the retinal tissue of USP21 knockout mice and wild-type mice.These data showed that dihydropyrimidinase-related protein 2(DPYSL2),a microtubule binding protein,can interact with USP21.The interaction between USP21 and DPYSL2 was confirmed by in vitro and in vivo coimmunoprecipitation experiments using retinal tissue,RPE1 cell line,HEK293 T cell line and purified protein.Furthermore,we demonstrated that DPYSL2 was a substrate of USP21.USP21 stabilized DPYSL2 by deubiquitination through the K48 proteasome pathway.By designing a series of DPYSL2 deletion mutation and lysine point mutation plasmids,we identified the USP21 deubiquitination site on DPYSL2 is K293.This part of the study shows that USP21 regulates ciliary formation of photoreceptor cells by stabilizing the downstream substrate DPYSL2.We further investigated the effect of DPYSL2 deletion on the primary cilia development.DPYSL2 deletion prevents IFT140 from being recruited to the parent centriolum.The integrity and proper localization of IFT140 is essential for the formation of transition zone,which is the precondition for the development of primary cilia.Therefore,this part of the study indicates that USP21-DPYSL2 axis regulates the accurate assembly of the transition zone by regulating IFT140,and the retinopathy induced by USP21 knockout is a series of pathological changes caused by the functional defects in the transition zone of photoreceptor cells’ cilia.In summary,USP21 plays a key role in the retinal development by promoting the cilia formation of the photoreceptor cells.It stabilize DPYSL2 via its deubiquitination activity.Then,the stabilized DPYSL2 ensure the successful recruitment of IFT140 to the ciliary transition zone of photoreceptor cells.The introduction of USP21-DPYSL2 axis not only shed light on the mechanisms in primary cilia regulation,but also provide a potential target for the treatment of retinopathy.