YTHDF2 phase separation promotes arsenic-induced keratinocyte transformation in a poly-m⁶A-dependent manner by inhibiting translational initiation of the key tumor suppressor PTEN

The phase separation of N⁶-methyladenosine (m⁶A) binding protein YTHDF2 plays a vital role in arsenic-induced skin damage, and YTHDF2 can bind to m⁶A-methylated mRNA of tumor suppressor PTEN. However, whether and how YTHDF2 phase separation regulates PTEN involved in arsenic-induced malignant transformation of keratinocytes remains blank. Here, we established arsenite-induced transformation models with stable expression of wild-type YTHDF2 or mutant YTHDF2 protein in vitro and in vivo. We found that the YTHDF2 protein underwent phase separation during arsenite-induced malignant transformation of keratinocytes, and YTHDF2 phase separation promoted the malignant phenotype of keratinocytes. Mechanically, YTHDF2 phase separation reduced PTEN protein levels, which in turn activated the pro-survival AKT signal. The binding of YTHDF2 to multiple m⁶A sites on PTEN mRNA drove YTHDF2 phase separation, inhibiting PTEN translation initiation and thus reducing PTEN protein levels. YTHDF2 phase separation recruited translation-initiation-factor kinase EIF2AK1 to phosphorylate eIF2α, thereby inhibiting translation initiation of poly-m⁶A-methylated PTEN mRNA. Furthermore, arsenite-induced oxidative stress triggered YTHDF2 phase separation by increasing m⁶A levels of PTEN mRNA. Our results demonstrated that YTHDF2 phase separation promotes arsenite-induced malignant transformation by inhibiting PTEN translation in a poly-m⁶A-dependent manner. This study sheds light on arsenic carcinogenicity from the novel aspect of m⁶A-mediated YTHDF2 phase separation.