AUF1 Can Recognize Cytoplasmic DNA Through RRM1/2 Domains

ObjectiveWe tended to identify new cytoplasmic DNA sensor and explore the interaction between AUF1 and cytoplasmic DNA.Method?1?Firstly,we use nuclear-cytosol fraction technology to isolate the nucleus and cytoplasm of MEF cells.Cytoplasmic DNA binding proteins were then separated by biotin-avidin purification system.The non-biotin labeled DNA and biotin labeled poly?dA?were used as control.We searched for the differential proteins using silver staining,and identified the proteins using mass spectrometry and complex-mass spectrometry technology.AUF1 was identified in both mass spectrometry.?2?We constructed the prokaryotic and eukaryotic expression vectors of p45^AUF1,GST-AUF1 and GFP-AUF1,which were incubated with biotin labeled dsISD,ssISD-F,ssISD-R,and poly?dA?and pull-downed with streptavidin beads.The interaction between p45^AUF1UF1 and ISD were determined by Western Blot.?3?We established AUF1 knockout HEK293T cell lines by CRISPR/Cas9technology.And the expression of AUF1 in knockout and wild type cell lines were determined by Western Blot.?4?We designed the gene specific cloning primers of AUF1 structural deletion,amplified the corresponding fragments with high-fidelity DNA polymerase,and finally ligate the fragments to pBOB-c-flag vector.?5?The flag-tagged p37,p40,p42 and p45 were transfected to HEK293T-AUF1K/O cells,and the localization of four subtypes was observed by immunofluorescence technique.?6?The flag-tagged p37,p40,p42,p45 and series of truncations recombinant plasmids were transfected into HEK293T-AUF1 K/O cells,and the cells werelysed and centrifuged after 48 h.We mixed supernatant liquid with biotin labeled ISD.The mixture was incubated with streptavidin beads.We further confirmed the binding between AUF1 and ISD by Western Blot.?7?The flag-tagged full length AUF1 and series of truncations recombinant plasmids were transfected into HEK293T-AUF1 K/O cells with GFP-AUF1 plasmids respectively,and the cells were collected,lysed and centrifuged after 48 h.The supernatant was then added with anti-flag beads.AUF1's self-interacion was determined by Western Blot.Result?1?AUF1 was identified as one of ISD binding proteins by mass spectrometry.?2?In vitro pull-down assay demonstrated that AUF1 interact with ISD,and its interaction with single-stranded ISD is stronger than that with double-stranded ISD.?3?AUF1 gene knockout HEK293T cell line was generated using CRISPR/Cas9technology.?4?p42 and P45 were mainly located in the nucleus,while p37 and p40 were mainly located in cytoplasm.?5?The four subtypes of AUF1 all binded to both dsISD and ssISD.?6?Deletion of RRM1or RRM2 can decrease the binding of AUF1 to ISD,While deletion of both RRM1 and RRM2 blocked the interaction of AUF1 and ISD.?7?AUF1 can bind to itself.?8?Deletion of RRM1and RRM2 made the binding between AUF1 itself disappear,while either RRM1or RRM2 did not.Conclusion?1?AUF1 binds to ISD.Exon 7 inhibits the binding between AUF1 and dsISD.?2?AUF1 binds to ISD through RRM1 and RRM2 domain.?3?AUF1 interacts with itself through RRM1 and RRM2 domain.