Mechanism Analysis Of Defected Primary Ciliogenesis Caused By ELMOD1 And ELMOD3 Deficiency

Objective:To explore the cellular function and mechanism of ELMOD1 and ELMOD3,regulators of ADP-ribosylation factors（ARFs）GTPase family,and to reveal the possible pathogenic mechanism of ELMOD1 and ELMOD3 mutations for further study.Methods:1.Elmod1 and/or Elmod3 null immortalized mouse embryonic fibroblast（MEF）lines(Elmod1^-/-,Elmod3^-/-and DKO)were generated and characterized using CRISPR-Cas9 gene knockout technology and DNA sequencing.2.Flow cytometry and immunofluorescence techniques were used to screen the cell cycle and submicroscopic structures such as nuclear,centriole and primary cilia of Elmod1^-/-,Elmod3^-/-and DKO MEF,compared with wild-type line（WT）as a control.After the abnormal phenotypes of knockout lines were identified,the rescue effect of these abnormal phenotypes was observed by transient transfection to make Elmod1^-/-and Elmod3^-/-express ELMOD1 or ELMOD3 exogenously.3.According to the abnormal phenotypes identified through phenotypic screening above,immunofluorescence technique was used to stain ELMOD1 and ELMOD3 proteins in MEFs and other related models,to identify their locations.4.Since ciliation defects were identified as the abnormal phenotypes in knockout lines through phenotypic screening above,their mechanisms were further explored.On the one hand,immunofluorescence technique was used to identify whether the expressions were altered of ciliary proteins required for ciliogenesis in Elmod1^-/-,Elmod3^-/-and DKO MEF;On the other hand,transient transfection were used to make Elmod1^-/-and Elmod3^-/-express fast-cycling mutants of several ARFs,respectively.Results of rescue experiments of abnormal ciliary phenotypes were observed to identify the ARF family members downstream of ELMOD1and ELMOD3 related to ciliogenesis.Results:1.Elmod1^-/-,Elmod3^-/-and DKO immortalized MEF lines were generated and characterized successfully.Two strains of each line were used for subsequent research.2.Results of phenotype screening suggested that no significant alterations of cell cycle and submicroscopic structures such as nuclear,mitochondria,Golgi-endosome,cytoskeleton and centriole of the knockout lines were observed except decreased ciliation,as compared with the WT.As for rescue experiment,decreased ciliation of Elmod1^-/-and Elmod3^-/-were significantly elevated by exogenous expression of ELMOD1 and ELMOD3 respectively.It proves that absence of ELMOD1or ELMOD3 leads to defects of ciliogenesis in MEFs.3.We failed in localization of ELMOD1 and ELMOD3 in MEFs.In the photoreceptor region of mouse retina,which often used in ciliary study,ELMOD1 and ELMOD3 were both located in the cilia-related region of connecting cilium,basal body and daughter centriole,which is consistent with their function in ciliogenesis.4.Decrease or absence of ARL13B,ARL3 and INPP5E in cilia,as well as accumulation of INPP5E in Golgi,were the major altered phenotypes of ciliary proteins of Elmod1^-/-,Elmod3^-/-and DKO.Regulatory factors in early stage of ciliogenesis（CEP164,CP110,CEP290）as well as intraflagellar transport complex（IFT140,IFT88）seemed normal in cilia-related region.5.Exogenous expression of ARL3 or ARL16 fast-cycling mutants could rescue the reduced ciliation and INPP5E accumulation in Golgi of Elmod1^-/-and Elmod3^-/-,which indicates that ARL3 and ARL16 are located downstream of ELMOD1 and ELMOD3 in ciliogenesis-related pathway.Conclusion:ELMOD1 and ELMOD3 are positive regulators necessary for primary cilia formation.The relevant molecular mechanism is that ELMOD1 and ELMOD3 can act as GTPase activating proteins to regulate the activities of ARL3 and ARL16,maintain the normal content of primary cilium-related proteins ARL13B,ARL3 and INPP5E in primary cilia and Golgi apparatus,and positively regulate the formation of primary cilia.Combining the importance of primary cilia to hearing and the cilia bundle defects in animal models of ELMOD1 and ELMOD3diseases,it is of great value to study the molecular mechanism of deafness caused by the deletion of ELMOD1 and ELMOD3 from the perspective of primary cilia.