Figure 2.

METTL3 expression regulates synthetic miniSINEUP activity

(A) m⁶A-RIP qPCR analysis. miniSINEUP-DJ-1 was transfected in shCtrl and shMETTL3 A549 cells. CREBBP and SON mRNA were used as endogenous positive controls, while HPRT1 mRNA was used as a negative control. Eluates from IgG immunoprecipitation were used as negative controls. IVT EGFP mRNA was spiked in total RNA extract to assess the specificity of the immunoprecipitation reaction. Data are mean ± SEM and are relative to n = 3 independent experiments. p values are calculated using two-way ANOVA and Šidák’s multiple-comparisons test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. (B) Protein expression analysis. miniSINEUP-DJ-1 activity was assessed using western blotting with anti-METTL3 and anti-DJ-1 antibodies in A549 shCtrl and shMETTL3 cells. ΔBD (miniSINEUP-DJ-1 deprived of BD) was used as a negative control in each condition. One representative experiment is shown (left). SINEUP activity was calculated as an increase in protein quantities relative to the negative control (ΔBD) for each condition. First, band intensity was normalized to the relative β-actin band. Then, fold change values for miniSINEUP-DJ-1-transfected samples were calculated normalizing on negative control-transfected cells for each cell line (dotted line) and reported in the reassuming graph (right). Data indicate single replicate values and mean ± SEM relative to n = 3 independent experiments. p values are calculated using two-way ANOVA with Šidák’s multiple comparison. ∗p < 0.05 and ∗∗p < 0.01.