Fig. 2
Mean Relative enrichment of target transcripts in 1ry NK cultured cells. To validate the bioinformatics results, MeRIP-qPCR was performed on expanded 1ry cultured NK cells from 3 control subjects (n = 3). Relative mRNA m6A quantification was conducted by analyzing the m6A level in target gene transcripts within the immunoprecipitated (IP) fraction, normalized against the non-immunoprecipitated (non-IPed)/input control RNA. The enrichment in the input control RNA was set to one. CT values were normalized to an exogenously added m6A-modified control (Gaussia luciferase), provided in the EpiMark® N6-methyladenosine Enrichment Kit (NEB, MA, USA). This RNA m6A-modified control was spiked into the samples before RNA fragmentation to monitor m6A enrichment. Compared to the input control, enrichment in the IP fraction of NKG2D was 2.3 folds higher (P = 0.0098, q = 0.0013), and the PI3K was fourfold higher (P & q =  < 0.0001), indicating that the m6A consensus motifs are significantly enriched in the 3’UTR of NKG2D and PI3K. PRF1 exhibited only a slight increase in m6A enrichment within the IP fraction, measuring 1.2-fold higher (P = 0.5853, q = 0.0683) than the input control fraction, but this difference was not statistically significant. In contrast, several transcripts showed significantly lower m6A levels in the IP fraction relative to the input control. VAV1 was 3.3-fold lower (P = 0.0037, q = 0.0006), PAK1 was 2.9-fold lower (P & q < 0.0001), ERK2 was 24-fold lower (P & q < 0.0001), GZMH was 3.5-fold lower (P & q < 0.0001), FASL was 11-fold lower (P & q < 0.0001), and TRAIL was twofold lower (P = 0.0007, q = 0.0001). The significantly lower m6A levels in the IP fraction for VAV1, PAK1, TRAIL, ERK2, FASL, and GZMH suggest that these transcripts are minimally methylated in this region. Statistical analysis was performed using a one-sample t-test to determine whether the mean enrichment in the IP fractions was statistically different from the input control, which was set to one