Fig. 3: Experimental validation of intron repression on m6A deposition.
a Illustration of the minigene experiment, using Lrp12 and Gne as two model genes. The minigene contained two exons and a 200 nt intervening mini-intron (details in Methods), and the first exon was in-frame fused to AcGFP1. Constructs were transfected into HEK293T cells, and m6A signal was quantified by SELECT method. b The pre-mRNA splicing of mini-intron in minigene of Lrp12 or Gne was validated by RT-PCR in HEK293T cells. At least three independent experiments were performed. c, d The bar plot of relative m6A level for detecting the m6A sites in mRNA. The constructs of minigenes were shown, and RAC sites in Lrp12 (c) or Gne (d) were marked as pink lines. Data were presented as mean ± SD, the P-values were calculated by two-sided Student’s t-test (p = 0.0065, 0.00013, 0.004, 0.011 for A1, A2, A3, A5 in Lrp12, p = 0.013, 0.005, 0.0008, 0.014, 0.003, 0.009 for A1, A4, A5, A6, A7, A11 in Gne. Significance: ***p < 0.001, **p < 0.01, *p < 0.05. n = 3 or 2 biological independent samples in (c) or (d)). The RAC site showed agreed increased m6A signal in both iM6A modeling and experimental validation was marked by blue box. e The dot plot of the experimental determined relative m6A level change for each RAC site in mRNA (19 sites in total). Relative m6A level change was calculated for the relative m6A level of each site between intron-containing and intron-deletion mRNAs. The mean value (0.264) was shown as the dotted pink line, and P-value was calculated by one-sample one-sided t-test for the increase vs. no change. f, g mRNA decay plotted as a function of time. The normalized levels of minigene mRNA at 0 h were set to 100%. The y axis represents the log value of mRNA remaining level. The P-values were calculated by two-sided Student’s t-test (p = 5.4e−6 in (f) for Lrp12, p = 0.0026 in (g) for Gne. Significance: ***p < 0.001, **p < 0.01).
