Research Paper Volume 13, Issue 19 pp 23361—23375

piRNA-36741 regulates BMP2-mediated osteoblast differentiation via METTL3 controlled m6A modification

piR-36741 bound to METTL3 to regulate the m6A activity of METTL3. (A) RNA pull-down assay was performed using bio-NC or bio-piR-36741 to verify the binding of piR-36741 and METTL3. (B–F) RIP assay was carried out to confirm the binding of piR-36741 with METTL3, METTL14, WTAP, FTO and ALKBH5. (G) Biotin-based RNA pull-down assay was used to detect the specific binding of piR-36741 with PIWIL4. (H) RIP assay was performed to verify the binding piR-36741 with PIWIL4. (I) BMSCs were transfected with METTL3 and PIWIL4 overexpression vectors, and co-IP was carried out using specific antibodies, and the immunocomplex were purified and subjected to Western blotting analysis using METTL3 or PIWIL4 antibodies. (J) RNA pull-down assay was used to confirm the binding ability of purified PIWIL4 or METTL3 proteins with biotin-labelled piR-36741. (K, L) BMSCs were transfected with mimic-piR-36741, antagomir-piR-36741 or respective controls for 24 h, and the mRNA and protein levels of METTL3 were determined. (M, N) m6A levels of BMSCs with or without piR-36741/PIWIL4 overexpression were detected by using the EpiQuik™ m6A RNA methylation quantification kit. N = 5 in each group. *P **P

Figure 3. piR-36741 bound to METTL3 to regulate the m6A activity of METTL3. (A) RNA pull-down assay was performed using bio-NC or bio-piR-36741 to verify the binding of piR-36741 and METTL3. (BF) RIP assay was carried out to confirm the binding of piR-36741 with METTL3, METTL14, WTAP, FTO and ALKBH5. (G) Biotin-based RNA pull-down assay was used to detect the specific binding of piR-36741 with PIWIL4. (H) RIP assay was performed to verify the binding piR-36741 with PIWIL4. (I) BMSCs were transfected with METTL3 and PIWIL4 overexpression vectors, and co-IP was carried out using specific antibodies, and the immunocomplex were purified and subjected to Western blotting analysis using METTL3 or PIWIL4 antibodies. (J) RNA pull-down assay was used to confirm the binding ability of purified PIWIL4 or METTL3 proteins with biotin-labelled piR-36741. (K, L) BMSCs were transfected with mimic-piR-36741, antagomir-piR-36741 or respective controls for 24 h, and the mRNA and protein levels of METTL3 were determined. (M, N) m6A levels of BMSCs with or without piR-36741/PIWIL4 overexpression were detected by using the EpiQuik™ m6A RNA methylation quantification kit. N = 5 in each group. *P < 0.05, **P < 0.01. Each test was independently repeated at least three times.