Fig. 4: N6-methyladenosine stabilizes Gys2 mRNA in an IGF2BP2-dependent manner.

a qRT-PCR assay of relative Gys2 mRNA level (β-actin as reference) in siControl and indicated siRNAs. n = 3 independent experiments. **p < 0.01 comparing with IGF2BP2:siControl. b qRT-PCR assay of Gys2 mRNA stability (β-actin as reference) in siControl and siIGF2BP2 Hepa1-6 cells treated with actinomycin D (Act D) at the indicated times. n = 3 independent experiments. *p = 0.022 at hour 2; p = 0.039 at hour 4. c enrichment of Gys2 mRNA in mettl3-HET or cKO hepatocytes by IGF2BP2-RIP-qPCR. n = 3 independent experiments. **p < 0.01 comparing with IgG:HET; ^##p < 0.01 comparing with IGF2BP2:HET. d Two conserved candidate m6A sites were predicted by SRAMP (http://www.cuilab.cn/sramp), according to MeRIP-seq data. Mutant #1 and #2 were constructs with “A to U” mutation at different predicted sites. Luciferase (Luc) or Flag-tag were fused in N terminal of each construct. qP1 and qP2 were specific qRT-PCR primers to detect exogenous WT or Mutant Flag-Gys2 mRNAs. e, f Flag-Gys2-CDS WT or indicated Mut construct and GFP overexpression constructs were transfected into Hepa1-6 cells for 48 h. Exogenous constructs expression levels were measured by qRT-PCR (e) and western blotting (f). GFP was reference in both assays. In figure e, n = 3 independent experiments. ns, not significant; *p < 0.05. The experiment in f was repeated independently with similar results at least 3 times. g pmirGLO Luc-Gys2-WT or indicated Mut construct was transfected into HEK-293T cells for 48 h. Relative luciferase activity (Fluc/Rluc) was tested by DLR assay. n = 3 independent experiments. ns, not significant; **p < 0.01. h enrichment of Flag-Gys2-CDS WT in siControl or siIGF2BP2 Hepa1-6 cells by IGF2BP2-RIP-qPCR. n = 3 independent experiments. **p = 0.007. For figure a–c, e, g, h, data are presented as mean values +/− SEM. For figure a, c, two-way ANOVA was performed to determine a difference among each two columns. For figure b, h, two-sided Student’s t test was performed to determine a difference between siControl and siIGF2BP2. For figure e, g, one-way ANOVA was performed to determine a difference between each column. Source data are provided as a Source data file.