Cancer-associated SF3B1 Mutations Affect The Interaction Between SF3B1 And DDX46

Backgroud: SF3B1 is an important component of U2 snRNP,which is required for the assembling of U2 snRNP and the stabilization of the interaction between U2 snRNP and branch point sequences(BPS).Mutations in RNA splicing factors have been detected in a variety of cancers.Among them,SF3B1 is the most frequently mutatated splicing factor.Furthermore,SF3B1 mutations res?lt in abnormal RNA splicing at the 3' splice site.DDX46 is a member of the DEAD-box RNA helicase family,which participate in pre-mRNA splicing by reg?lating the conformation of U2 snRNP.Previous studies show that Prp5 p,the yeast homologous gene of DDX46,directly interacts with Hsh155 p,the yeast homologous gene of SF3B1.Objective: This study aims to investigate whether cancer-associated SF3B1 mutations affect the interaction between SF3B1 and DDX46 and whether the altered interaction between SF3B1 and DDX46 causes abnormal RNA splicing in 293 T cells.Method: The plasmids harboring FLAG-SF3B1-WT or FLAG-SF3B1-K700 E were constructed and transfected into 293 T cells;Co-immunoprecipitation was used to enrich proteins that interacted with FLAG-SF3B1-WT or FLAG-SF3B1-K700 E using FLAG antibody,and immunoprecipitation and western blot were used to detect the interactions between DDX46 and SF3B1.Abnormal splicing affected by knockdowns or overexpression of DDX46 were detected by Taqman PCR.The interactions beween DDX46 and 9 others mutatant SF3B1 were examined by western blot,while the abnormal RNA splicing induced by there mutant SF3B1 was examined by Taqman PCR.Res?lts: SF3B1-K700 E mutation caused decreased interaction between SF3B1 and DDX46.While erexpression of DDX46 increases the interaction between DDX46 and K700 E mutated SF3B1,it does not inhibit the SF3B1-K700 E mutation induced abnormal RNA splicing.Knockdown of DDX46 does not have the same effect on RNA abnormal splicing as SF3B1 mutation.Although 9 other types of mutant SF3B1 also demonstrate decreased interaction between SF3B1 and DDX46,the decreased interaction between SF3B1 and DDX46 dose not have a lineav correlation with the abnormal RNA splicing induced by these mutant SF3B1.Conclusion: Cancer-associated SF3B1 mutants caused significantly reduced interactions between SF3B1 and DDX46 in 293 T cells,but the reduced interactions between mutant SF3B1 and DDX46 is not the molec?lar mechanism that causes SF3B1 mutation induced abnormal RNA splicing.