The Functional Study Of Retinal Degeneration-Related Genes WTAP And EMC6 In Photoreceptor Cells

Retinitis pigmentosa（RP）is a genetic eye disease caused by the degeneration of photoreceptor cells in the retina.Symptoms in RP patients typically manifest as night blindness and progressive narrowing of the visual field,worsening over time to tunnel vision and eventually leading to blindness.In China,the number of RP patients exceeds300,000,causing significant impact on both the families of the patients and society.Due to the clinical and genetic heterogeneity of RP,diagnosis and treatment remain substantial challenges.Consequently,the identification of novel pathogenic genes for RP,elucidation of the disease mechanisms,and the development of efficacious treatments constitute imperative objectives at present.The exploration of new pathogenic genes for RP can be pursued through two primary methodologies:firstly,by analyzing potential disease-associated genes in RP families where genetic causes remain unexplained,and secondly,by investigating the relationship between pathogenic genes identified in families with established visual impairments and RP.To this point,approximately 90 genes implicated in RP have been cataloged.However,mutations in these genes account for only about 70%of RP cases,leaving nearly 30%with etiologies that are still unidentified.It is posited that the majority of these cases might result from non-traditional genetic mutations,such as those involving epigenetic modifications.Accordingly,this study has concentrated on the most prevalent form of m RNA modification in eukaryotes,N6-methyladenosine（m⁶A）,and selected the core subunit of the methyltransferase complex,Wilms Tumor 1Associated Protein（WTAP）,for in-depth examination.Furthermore,mutations in the EMC1 gene have been detected in numerous families afflicted with visual function impairments,which implies a potential role for EMC in the retinal context.Consequently,the core subunit of EMC,ER Membrane Protein Complex 6（EMC6）,was also selected for analysis.This dissertation rigorously investigates the roles and mechanisms of WTAP and EMC6 within photoreceptor cells,as elaborated subsequently:1.Study on the Function of WTAP in Photoreceptor Cells:m⁶A modification,as one of the most common epigenetic modifications in eukaryotic m RNA,has been shown to play a role in the development and function of the mammalian nervous system by regulating m RNA synthesis and degradation.However,the specific role of m⁶A modification in retinal photoreceptor cells is not yet clear.In this study,focusing on the core subunit of the m⁶A methyltransferase complex,WTAP,we created mouse models with specific knockout of the Wtap gene in photoreceptor cells(Wtap^RKO and Wtap^CKOmice)using Rod-Cre and Cone-Cre mice.The data indicate that the absence of Wtap in rod cells led to a series of RP-like phenotypes in Wtap ^RKO mice,including progressive retinal thinning,impaired visual function,fundus pigment deposition,shortened OS,and reduced expression of membrane proteins.Similarly,defects in Wtap in cone cells led to the gradual degeneration of cone opsins.Moreover,knocking out Wtap significantly reduced the expression levels of other subunits of the m⁶A methyltransferase complex and m⁶A methylation levels in the retina.Multi-omics analysis revealed that Wtap deficiency leads to reduced m⁶A modification in the 3’UTR regions of key genes Pde6b,Reep6,and Rdh12 in rod cells,causing decreased translation efficiency,leading to significantly reduced protein expression and ultimately causing the gradual death of rod cells.Overexpression of WTAP protein in photoreceptor cells significantly improved the retinal lesion phenotype in Wtap^RKO mice,highlighting the potential of WTAP as a therapeutic target.Overall,Wtap influences the expression of Pde6b,Reep6,and Rdh12through the regulation of m⁶A modification,ultimately leading to rod cell death.These findings underline the crucial role of WTAP as a core subunit of the m⁶A methyltransferase complex in maintaining photoreceptor cell function.2.Study on the Function of EMC6 in Photoreceptor Cells:EMC monitors the biosynthesis of membrane proteins with multiple transmembrane domains or tail anchorage（TA）.Although studies have shown that knocking out the Emc3 gene leads to mislocalization of rhodopsin（RHO）in mouse rod cells,the exact mechanism causing abnormal RHO transport remains unclear.This study is the first to construct mouse models specifically knocking out Emc6 in rod and cone cells(Emc6^RKO and Emc6^CKOmice).The absence of Emc6 in rod cells resulted in gradually shortened photoreceptor OS,impaired visual function,mislocalized and reduced expression of RHO,and significant proliferation of astrocytes in retinas.Additionally,Emc6^CKO mice exhibited gradual death of cone cells and abnormal localization of cone opsins.Proteomic analysis showed that the expression of several cilia-related proteins,especially ANO2 and TMEM67 with multiple transmembrane domains,was significantly downregulated in the retinas of Emc6^RKO mice.Further studies confirmed that the loss of Emc6 led to significant reductions in the expression of ANO2 and TMEM67.In Emc6^RKO mice,the cilia of rod cells were notably damaged,leading to the mislocalization of several key OS membrane proteins.The results indicate that Emc6 not only plays a key role in monitoring the synthesis of RHO but also affects the ciliary function of rod cells by regulating the synthesis of cilia-related membrane proteins ANO2 and TMEM67,resulting in the mislocalization of disc proteins.These factors collectively lead to a significant reduction in the transport of disc proteins to the OS,ultimately causing OS degeneration and photoreceptor cell death.In conclusion,this dissertation clarifies the critical functions of WTAP and EMC6in maintaining photoreceptor cell function,offering potential directions for the development of therapeutic strategies for retinal diseases.