Fig. 4.

m⁶A motifs within the 3′UTR of ITGA6 promote the translation of ITGA6 mRNA.

a psiCHECK™-2 luciferase reporter constructs containing fragments of the human ITGA6 3′UTR with 4 putative wild-type m⁶A motifs (WT) or mutant (A-to-T mutation) m⁶A sites (MUT1, MUT2, MUT3, MUT4, MUT1 + 2, MUT1 + 2 + 3, or MUT1 + 2 + 3 + 4) are shown. The position of the m⁶A sites (96, 111, 195, and 233) is numbered relative to the first nucleotide of the 3′UTR. b Relative luciferase activity of the psiCHECK™-2-ITGA6 3′UTR with either wild-type (WT) or single mutation of m⁶A sites 1, 2, 3, or 4 (MUT1, MUT2, MUT3, or MUT4) in METTL3-overexpressing 293 T cells. Renilla luciferase activity was measured and normalized to firefly luciferase activity. c Relative luciferase activity of psiCHECK™-2-ITGA6 3′UTR with either wild-type (WT) or mutant m⁶A sites (MUT1, MUT1 + 2, MUT1 + 2 + 3, or MUT1 + 2 + 3 + 4) in control and METTL3-overexpressing 293 T cells. Renilla luciferase activity was measured and normalized to firefly luciferase activity. d m⁶A enrichment in the psiCHECK™-2-ITGA6 mRNA 3′UTR was validated by MeRIP-qPCR in control and OE-METTL3 293T cells after transfection of the WT and MUT1 + 2 + 3 + 4 plasmids. The primer covers the junction between Renilla Luc and the ITGA6 3′UTR. e Renilla luciferase activity was detected in vitro using the Flexi Rabbit Reticulocyte Lysate System. Renilla luciferase reporter mRNAs with the ITGA6 3′UTR (WT, MUT1, MUT1 + 2, MUT1 + 2 + 3, or MUT1 + 2 + 3 + 4) were transcribed in vitro in the absence or presence of m⁶A. All bar plot data are the means ± SEMs of three independent experiments except in (c), where the error bars denote the SDs of technical triplicates. *p < .05, **p < .01, ***p < .001, ****p < .0001. (Student's t-test, one-way ANOVA, Dunnett ‘s test).