Figure 4. Selective 5′UTR m⁶A modification mediates cap-independent translation.

a, MEF cells transfected with Fluc mRNA reporters were subject to heat shock treatment and the Fluc activity was measured by real-time luminometry. Fluc activities were quantified and normalized to the one containing normal As. b, Constructs expressing Fluc reporter with Hsp70 5′UTR or the one with A103C mutation are depicted on the top. Fluc activities in transfected MEF cells were quantified and normalized to the control containing normal A without stress. c, Fluc mRNAs bearing Hsp70 5′UTR with a single m⁶A site were constructed using sequential splint ligation. After in vitro translation in rabbit reticulate lysates, Fluc activities were quantified and normalized to the control lacking m⁶A. Error bars, mean ± s.e.m.; * p < 0.05, unpaired two-tailed t-test; n=3, biological replicates (a, b and c). d, A proposed model for dynamic m⁶A 5′UTR methylation in response to stress and its role in cap-independent translation. Under the normal growth condition, nuclear FTO demethylates the 5′UTR m⁶A from nascent transcripts and the matured transcripts are translated via a cap-dependent mechanism. Under stress conditions, nuclear localization of YTHDF2 protects the 5′UTR of stress-induced transcripts from demethylation. With enhanced 5′UTR methylation, these transcripts are selectively translated via a cap-independent mechanism.