Characteristics Of The Interaction Between Stk38 And Rbm24 In Cardiomyocytes

AimsThe molecular mechanism of Rbm24 in cardiomyocytes is not fully understood.Our studies are to find the proteins that could bind to Rbm24 and explore their interaction and mechanism,hoping to find the new data and theoretical basis for the sarcomere assembly and sarcomere stability Rbm24 may participated in cardiomyocytes.MethodsThe proteins that bind to Rbm24 were searched by pull down assay and mass spectrometry analysis,and confirmed the information of this protein.The expression vector of this protein was constructed and then the interaction between it and Rbm24 was confirmed by co-immunoprecipitation after we co-transfect them into 293FT cell lines.We also detected that whether they can interact with each other in the endogenous expression by using H9C2 and HL-1 cell lines which both of them could express in.Then we confirmed that whether their interaction is dependent on RNA by degragate RNA using RnaseA treatment.Finally,immunofluorescence assay was used to detect the expression of this protein in H9C2 cells and to observe whether it can coexpression with Rbm24 in H9C2 cells.The cell lines,which the protein interacting with Rbm24 was knowdown by lentivirus infection,was constructed subsequently,and RT-qPCR and Western Blot analysis were used to detect the expression of Rbm24 and Rbm24-regulated myocardium-specific genes.ResultsThe protein Stk38(Serine/Threonine Kinase 3 8),which can interact with Rbm24,was found using pull-down assay and mass spectrometry analysis in H9C2-Flag-Rbm24 cell lines.We determined that Stk38 can interact with Rbm24 through co-immunoprecipitation.Futher immunoprecipitating experiments in H9C2 cells and HL-1 cells showed that Stk38 binds only to full-length Rbm24 protein and their combination is direct,which do not depend on RNA.After that we constructed a knockdown Stk38 cardiomyocyte line HL-1-shStk38.Using RT-qPCR and Western Blot analysis,we found that Stk38 almost did not affect the expression of Rbm24’mRNA but decreased the protein expression of Rbm24 after knockdown Stk38.Interestingly,the mRNA and protein levels of several myocardium sarcomere genes regulated by Rbm24 showed the same trend as Rbm24.ConclusionsStk38 can bind to Rbm24,which only need its full-length forms,directly.And Stk38 regulates the protein expression of Rbm24 but scarcely influences the mRNA expression of Rbm24.The expression both in mRNA and protein levels of Rbm24-regulated myocardium carcomere genes are the same as Rbm24.