Fig. 2
DNMT3A/AGO4 methylates miRNA. a siRNA against DNMT3A or AGO4 decreased the methylation level of miRNA-181a-5p. Expression of miRNA-181a-5p was assessed by qPCR and 5mC-IPed estimated the methylation level of the miRNA under the different siRNA tested. b Western blot experiments after immunoprecipitation using the Catch and Release® v2.0 Reversible Immunoprecipitation System (Milipore, France) and 4 μg IgG (negative control) or DNMT3A antibodies. Analysis of the DNMT3A or AGO4 expression after cell transfection with the indicated antibody. The Pro-Ject Protein Transfection Reagent kit (Thermo Scientific, France) was used to deliver antibodies in living cells according to the manufacturer’s instructions. IgG (10 μg) was used as a negative antibody control and α AGO41–164, (10 μg, Active Motif (AM39855), France) an antibody directed against the 1–164 amino acid region of AGO4, was used as to block the DNMT3A/AGO4 interaction. C. Proximity Ligation In Situ Assays were performed to investigate the interaction or close proximity between DNMT3A and AGO4 in U87 cells treated with control siRNA, siRNA targeting DNMT3 or AGO4. Red dots represent DNMT3A/AGO4 interactions. Nuclei are stained with DAPI (blue). The quantification of DNMT3A/AGO4 interactions (average ± standard deviation) was performed in 30 cells in three independent experiments. d Western blot experiments were performed after His-pull-down assay using His-DNMT3A and GST-AGO4 as bait and prey proteins respectively. IgG (4 μg) was used as a negative antibody control and αAGO41–164 (4 μg, Active Motif (AM39855), France), an antibody directed against the 1–164 amino acid region of AGO4, was used as blocker of DNMT3A/AGO4 interaction. e DNMT Magnetic Beads (DMB) Assay using DNMT3A and/or AGO4 (300 nM), AdoMet (900 nM), synthetic double-stranded DNA oligonucleotides (ds DNA) or synthetic miRNA. The mean values of triplicate experiments are presented with standard deviation error bars. IgG (4 μg) was used as a negative antibody control and αAGO41–164 (4 μg, Active Motif (AM39855), France) to block the DNMT3A/AGO4 interaction. f Cytosine-methylation profile of miRNAs immunoprecipitated by an anti-5methylcytosine. The graph illustrates the cytosine-methylation level of the 18 miRNA identified as methylated via the miRIP-5mC/Array method (according to Fig. 1e) in U87 cells treated or not (blue circle) with siRNA-DNMT3A (red circle), siRNA-AGO4 (green circle) and αAGO41–164 (purple circle). g 5mC quantification using ELISA in 100 ng of miRNA from cells treated or not with the indicated antibodies. The Pro-Ject Protein Transfection Reagent kit (Thermo Scientific, France) was used to deliver antibodies to living cells according to the manufacturer’s instructions. IgG (10 μg) was used as a negative antibody control and αAGO41–164 (10 μg, Active Motif (AM39855), France) was used to block the DNMT3A/AGO4 interaction. Mean values of triplicate experiments presented with standard deviation error bars