Fig. 7.

CircMRPS35 Acts as a Modular Scaffold to Recruit KAT7 to the Promoters of FOXO1 and FOXO3a Gene. a The experimental design for pull-down assay. RNA pull-down was performed using biotinylated circMRPS35 probe, followed by mass spectrometry. b RNA pull-down assay followed by western blot for candidate proteins KAT7, TAF15 and BTF3 in MGC803 cells. c RIP assay for the binding of three candidate proteins with circMRPS35. RIP was performed using KAT7, TAF15 and BTF3 antibodies, followed by qRT-PCR assay for circMRPS35 expression in MGC803 cells. d FISH for circMRPS35 and IF for KAT7 in MGC803 cells. The profiles of colocalization were also provided. Scale bar, 20 μm. e The molecular docking of the interaction between circMRPS35 fragment (back-splice sites, sequence: AAGACGGA) (marked stick) and KAT7 (shown as green). f The design of the truncated KAT7 expression plasmids. g RNA pull-down assay after transfection of wild type and truncated KAT7 expression plasmids using biotin-labelled oligo or circMRPS35 probes in MGC803 cells. h-i ChIP assay for H4K5ac level in FOXO1 h and FOXO3a i promoter regions. Final DNA extractions were PCR amplified using primers that cover P1 (− 839~ − 1028), P2 (− 1865~ − 2045) in FOXO1 promoter and p1 (− 1560~ − 1739), p2 (− 1976~ − 2155), p3 (− 3159~ − 3347) in FOXO3a promoter. j FISH for circMRPS35 and IF for H4K5ac in MGC803 cells. The profiles of colocalization are also provided. Scale bar, 20 μm. k RNA pull-down assay followed by western blot for H4K5ac expression in MGC803 cells. l RIP assay for the binding of H4K5ac with circMRPS35. RIP was performed using H4K5ac antibody, followed by qRT-PCR for circMRPS35 expression in MGC803 cells. m Colocalization analysis of circMRPS35, KAT7 and H4K5ac by super high resolution microscopy in MGC803 cells. Scale bar, 10 μm. n Z-DOCK of prediction of the trimer complex structure of circMRPS35 fragment (marked stick), H4K5ac (marked blue cartoon), and KAT7 (marked green cartoon). The significant binding interaction was marked by a red line. o ChIRP assay for the binding of circMRPS35 to FOXO1 and FOXO3a promoters in MGC803 cells. LacZ was a negative control. The PCR primers covered P2 (− 1865~ − 2045) in FOXO1 promoter and p1 (− 1560~ − 1739) in FOXO3a promoter, respectively. p-q ChIP assay for H4K5ac levels in FOXO1 p and FOXO3a q promoters after KAT7 knockdown. SGC7901 cells were transfected with circMRPS35 or control plasmids, together with KAT7 siRNA and the corresponding control. Cells were harvested for ChIP assay 48 h later. r qRT-PCR for FOXO1 and FOXO3a mRNA expression in SGC7901 cells treated as in p. s Western blot assay for KAT7, FOXO1 and FOXO3a protein levels in cells treated as in p. Cells were harvested 48 h later for Western blot analysis. *P < 0.05, **P < 0.01, ***P < 0.001