Research On The Mechanism Of RNA Editing Enzyme ADAR1 In Maintaining The Genome Stability Of Hematopoietic Stem And Progenitor Cells

Objective:Human genomic DNA is threatened to variety degrees every day for varying reasons.Prompt and correct repair of damage is essential for maintaining genomic stability.To deal with these DNA damages,our bodies have evolved a sophisticated and precise system.These include three phosphatidylinositol 3-kinase-associated kinases(PIKKs):ATM,ATR,and DNA-PK.ATM binds to MRN(MRE11-RAD50-NBS1)complex and DNA-PK binds to Ku(Ku70-Ku80)complex when DNA double-stranded damage occurs.When there is single-stranded DNA(ss DNA)exposure in cells due to replication stress or other reasons,the RPA(RPA70-RPA32-RPA14)complex will bind to the exposed ss DNA,and ATR-ATRIP will bind to the ss DNA-RPA complex.So far,there is no evidence that ATR can be directly activated by the ss DNA-RPA complex.More literatures have shown that ATR activation requires the assistance of ATR activation proteins TOPBP1 and ETAA1.But the exact mechanism is unclear.When intracellular replication stress existde,if it cannot be resolved in time,the process of transcription and replication is prone to collide,thus leading to the formation of R-loop.ADAR1 is an RNA editing enzyme with A-to-I editing activity for double-stranded RNA(ds RNA).At present,most of the studies on ADAR1 focus on the enzyme activity of RNA editing and its function in the immune system,and little is known about the function of ADAR1 in other fields.Our studies have shown that ADAR1 has the function of regulating the activation of ATR signaling pathway and regulating the Rloop process.Methods:First,immunoaffinity purification and mass spectrometry were used to find the interaction proteome of ADAR1 in human body.By analyzing the protein functions in the interaction group,we could guess in which fields there might be new functions of ADAR1 that have not been discovered.After the proteins of interest were found,the accuracy of the results of mass spectrometry was verified by internal and external immunoprecipitation experiments.We found TOPBP1 which related to ATR signaling pathway and DDX21 and DHX9 proteins related to R-loop formation.So we assume that ADAR1 is related to these two processes.Through a series of molecular experiments including Alkaline Comet Assay,Western Blot,Immunofluorescence Staining and DNA Fiber,we found that ADAR1 indeed has regulatory functions in the activation of ATR signaling pathway and the formation of R-loop.Through a series of phenotypic experiments such as Drug Sensitivity Test,Clone Formation Test and so on,as well as further animal experiments such as the construction of animal model of Adar1 gene knockout mouse and the extraction of hematopoietic progenitor cells,the accuracy of our molecular experiments was confirmed.Results:1.The proliferation and differentiation ability of hematopoietic progenitor cells in mice with Adar1 gene deletion was significantly reduced,and the level of γH2AX foci was significantly increased.2.ADAR1 interacts with TOPBP1 in vivo,and the interaction depends on the KKXXK motif in the three RNA binding domains.3.ADAR1 is involved in regulating the activation of ATR signaling pathway.4.ADAR1 regulates the activation of ATR signaling pathway by affecting the recruitment of TOPBP1,and this function is also dependent on the KKXXK motif.5.ADAR1 is involved in the regulation of intracellular R-loop level.6.ADRA1 does not recruit to replicate stress site,but dissociates from the site.Conclusion:According to the experiments,we draw the following conclusions:On the one hand,there is an interaction between ADAR1 and TOPBP1 in vivo,and the interaction is dependent on KKXXK motif.Moreover,ADAR1 affects the activation of ATR signaling pathway by regulating the recruitment of TOPBP1.This suggests that ADAR1 plays a role in DNA damage in response to replication stress.On the other hand,ADAR1 is involved in regulating the level of R-loop.These functions lead to increased levels of DNA damage in cells with ADAR1 deletion,thus affecting genomic stability.And we did find increased DNA damage in the hematopoietic progenitor cells of Adar1-deficient mice.The above studies provide a new idea for us to understand the self-renewal of hematopoietic progenitor cells and the maintenance of the stability of hematopoietic system。...