The RNA demethylases ALKBH5 and FTO regulate translation of the ATF4 mRNA in sorafenib-treated hepatocarcinoma cells

Translation is one of the main gene expression steps targeted by cellular stress, commonly refereed as translational stress, which includes treatment with anticancer drugs. While translational stress blocks translation initiation of bulk mRNAs, it allows translation of a specific set of mRNAs known as short upstream open reading frames (uORFs)-mRNAs. Among these, ATF4 mRNA encodes a transcription factor that reprograms gene expression during various cellular stress towards functions required for cell response to stress. Stress-induced ATF4 mRNA translation occurs via a specialised mode that relies on the presence of uORFs upstream to the main ATF4 ORF. However, mechanisms regulating ATF4 mRNA translation, particularly towards chemoresistance, remained limited. Here, we report a role of both ALKBH5 and FTO, the two RNA demethylating enzymes in promoting translation of ATF4 mRNA in liver cancer Hep3B cells treated with sorafenib, a stress inducer used in chemotherapy. Depletion experiments confirmed that both enzymes are required for inducing ATF4 mRNA translation, while polyribosome assays coupled to RT-qPCR indicated that this induction of ATF4 mRNA translation occurs at its initiation step. Using in vitro methylation assays, we found that ALKBH5 is required for the inhibition of the methylation of a reporter ATF4 mRNA at a conserved adenosine (A235) site located at its uORF2, suggesting that ALKBH5-mediated translation of ATF4 mRNA involves demethylation of its A235. Preventing methylation of A235 by introducing an A/G mutation into the ATF4 mRNA reporter renders that reporter insensitive to ALKBH5 depletion, supporting the role of ALKBH5 demethylation activity in translation. Finally, targeting either ALKBH5 or FTO sensitizes Hep3B to sorafenib-induced cell death, contributing to their resistance. We concluded that ALKBH5 and FTO are novel factors that promote resistance to sorafenib treatment, in part by mediating translation of ATF4 mRNA.