A Study On The Interaction Between AGO2 And DDX21

AGO2 also termed as EIF2C2,is a member of the Argonaute family of proteins.AGO2 is the core element of the RISC,and AGO2 can initiate the degradation of the target m RNA through gene silencing guided by si RNA or mi RNA.AGO2 can also repress target gene expression at the level of translation.AGO2 plays a critical role in mi RNA biogenesis.AGO2 has been found in nucleus,which engages in heterochromatinization,alternative splicing and DNA double-strand break repair by homologous recombination.By applying co-immunprecipitation with anti-AGO2 antibody couple to mass spectrometry,we determined that DDX21 acts as a potential AGO2 interacting protein.DDX21 is a member of the DEAD box family of helicases.DDX21 is a protein having RNA unwinding and RNA folding activities.DDX21 plays a critical role in multiple steps of ribosome biogenesis.DDX21 can also support the activation of transcription factor.This paper studied the interaction between AGO2 and DDX21 by using immunofluorescence technique,co-immunoprecipitation technique and GST-pulldown assay.These results of immunofluorescence and co-immunoprecipitation showed that AGO2 and DDX21 co-localized in the nuclei and they could interact with each other.Then,we used purified GST-tagged AGO2 and His-tagged DDX21 to perform GST-pulldown assay.The result showed that DDX21 binded with AGO2 indirectly in vitro.In order to identify the bridge of the interaction between AGO2 and DDX21,we added RNase A to the co-immunoprecipitaton system.The result showed that the interaction between AGO2 and DDX21 was weakened via RNase A.Therefore the interaction between AGO2 and DDX21 is associated with RNA.Next,we overexpressed AGO2 or DDX21 in the HeLa cell,and then we used Western Blot to detect the changes of protein level of DDX21 or AGO2.Those results showed that DDX21 could up-regulate AGO2 at the level of protein,however,AGO2 did not affect DDX21 at the level of protein.Moreover,we found that DDX21 could only up-regulate the protein level of nuclear AGO2.It was reported that AGO2 could affect the alternative splicing of SMN2.We overexpressed DDX21 in the HeLa cell first,and then used PCR to detect the alternative splicing change of SMN2.The result showed that overexpressed DDX21 affected the alternative splicing of SMN2.Taken together,this paper defined that there is an interaction between AGO2 and DDX21,and this interaction is associated with RNA.Further studies found that DDX21 could up-regulate nuclear AGO2 at the level of protein,and affect the alternative splicing influenced by AGO2.