Figure 5: Fos ecRNA interacts with DNA methyltransferases and blocks DNA methylation.
From: Extra-coding RNAs regulate neuronal DNA methylation dynamics
(a) Immunostaining reveals nuclear localization of DNMT1 and DNMT3a in neuronal cultures. Cell nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI), and neurons are marked by MAP2 (microtubule-associated protein 2). Scale bar, 50 μm. (b) Fos ecRNA/mRNA comparison in total neuronal lysate and nuclear fraction (separated during RNA-IP; n=4 per group; unpaired Student’s t-test, t6=6.301, P=0.007). (c) Fos ecRNA, but not mRNA, immunoprecipitates with anti-DNMT1 or DNMT3a antibodies but not control IgG (n=4–6 per group; ecRNA one-way ANOVA, F(2,15)=20.53, P<0.0001; Tukey’s post hoc test for individual comparisons). (d) Locations of synthetic RNA and DNA oligonucleotides used in mobility shift assays. (e) Electrophoretic mobility shift assay reveals only slight binding of ecRNA probes (1 nM) to recombinant DNMT1 (0.2 μM). RNA/DNMT complexes are evident as low-mobility band on native PAGE gel following incubation with DNMT protein. (f) Synthetic ecRNA probes (1 nM) bind recombinant DNMT3a/DNMT3l protein (0.2 μM). (g) Complete binding of synthetic ecRNA probes (1 nM) to truncated recombinant DNMT3a protein (0.2 μM) containing only catalytic domain (DNMT3a-CD). (h) Incubation of ecRNA-1 or ecDNA-1 probes (1 nM) with escalating concentrations of DNMT3a-CD (0.002–0.2 μM). (i) DNMT3a-CD binds equally to RNA and double-stranded DNA with the same primary sequence. Binding affinity (Kd values; derived from non-linear, one-site regression analysis of complete concentration curve) for RNA and dsDNA was not significantly different (n=2 replicates; comparison of Kd, F(1,22)=0.47, P=0.49). (j) Competition assay between ecRNA-1 (1 nM) and unlabelled ecDNA-1 probes (1–100 nM) shows intact RNA/DNMT3a-CD complexes even with 10-fold higher concentrations of DNA. (k) Quantification of dsDNA competition assay. (l) Co-incubation of DNMT3a/3l protein and the methyl donor SAM results in cytosine methylation at dsDNA (3.42 nM) from the Fos promoter. Methylation was significantly inhibited by addition of ecRNA (5.16 μM). For (l) n=4 per group, one-way ANOVA, F(4,15)=40.25, P<0.0001; Tukey’s post hoc test for individual comparisons. All data are expressed as mean±s.e.m. Individual comparisons, *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.
