Differential requirement of m ⁶ A reader proteins, IGF2BP2 and HNRNPA2B1 for the processing of N ⁶ -methyladenosine modified H19 lncRNA: Stability versus miR-675 biogenesis

H19, a lnc-pri-miRNA that encodes miR-675, is dysregulated in numerous cancers. However, the specific mechanisms underlying H19 processing, particularly miR-675 formation, remain unclear. Our study reveals that H19 is highly expressed and m ⁶ A modified in a METTL3-dependent manner in glioblastoma (GBM) and glioma stem cells (GSCs). Silencing METTL3 reduced both H19 and miR-675 levels, whereas overexpressing METTL3 promoted miR-675 processing without affecting H19 levels. Further, miR-675 derived from exogenously expressed H19 was affected considerably more in METTL3 silenced glioma cells compared to H19 levels, suggesting differential requirements in the processing of m ⁶ A modified H19 transcript. We demonstrate that H19 interacts with m ⁶ A reader proteins, IGF2BP2 and HNRNPA2B1, and silencing either reduced H19 and miR-675 levels. However, a high level of miR-675 seen in METTL3 overexpressing cells is severely affected in HNRNPA2B1-silenced compared to IGF2BP2-silenced glioma cells. Interestingly, IGF2BP2 silencing more significantly affected H19 stability from exogenous H19 construct, while HNRNPA2B1 silencing severely impacted miR-675 processing. Site-directed mutagenesis confirmed the presence of two m ⁶ A sites in the first exon of H19, with site #1 facilitating HNRNPA2B1 interaction to promote miR-675 processing. In contrast, the IGF2BP2 interaction is promoted by site #2, resulting in enhanced H19 stability. H19-METTL3-HNRNPA2B1-miR675 axis inhibited Calneuron 1 (CALN1), a known target of miR-675, to promote glioma cell migration. Notably, a low CALN1/high H19 predicted a poor prognosis in GBM patients and was further exacerbated by a high METTL3 or HNRNPA2B1 but not IGF2BP2 transcript levels. Thus, we found that the H19 transcript is highly expressed in GBM and m ⁶ A modified, and the m ⁶ A reader proteins, IGF2BP2 and HNRNPA2B1, regulate the H19 processing differently to promote glioma cell migration.