Post-transcriptional regulation of H19 by m⁶A reader proteins: H19 stability versus miR-675 biogenesis

While H19, a lnc-pri-miRNA that encodes miR-675, is known to be dysregulated in cancers, the mechanism governing its post-transcriptional regulation remains unclear. We found that H19 is highly expressed and m⁶A-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. Furthermore, 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 m⁶A-modified H19 transcripts. We demonstrate that H19 interacts with m⁶A reader proteins, IGF2BP2 and HNRNPA2B1, and silencing either of these proteins reduces 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 the exogenous H19 construct, while HNRNPA2B1 silencing severely impacted miR-675 processing. Of the two m⁶A sites identified, site #1 facilitated HNRNPA2B1 interaction to promote miR-675 processing, but site #2 enhanced IGF2BP2 interaction to increase H19 stability. H19-METTL3-HNRNPA2B1-miR-675 axis inhibited Calneuron 1 (CALN1), 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 H19 is a m⁶A modified transcript that is highly expressed in GBM, and the m⁶A reader proteins, IGF2BP2 and HNRNPA2B1, regulate H19 processing differently to promote glioma cell migration.