Figure 2. m⁶A within the 5′ UTR Enables Cap-Independent Translation of mRNA.

(A) 5′ UTR m⁶A permits mRNA translation without the need for the cap-binding protein eIF4E. In vitro translation was performed using a HeLa cell extract mixed with luciferase-encoding, capped mRNA containing either A or m⁶A. Protein production was measured by quantifying luciferase activity. Cap-dependent translation is observed from both methylated and unmethylated mRNAs in the presence of eIF4E. However, when eIF4E is absent, only the m⁶A-containing mRNA is translated (n = 4; mean ± SD; ***p < 0.0001).

(B) Presence of a 5′ cap analog is unable to abolish m⁶A-induced mRNA translation. Luciferase mRNAs were translated as in (A). 1 mM free cap analog (m7GpppG) was added to sequester cap-binding proteins. Addition of m7GpppG abolishes cap-dependent translation of unmethylated mRNA (left) but is unable to abolish the cap-independent translation induced by m⁶A (right). Levels of luciferase activity are shown relative to capped mRNA + 10 pmole eIF4E (n = 3; mean ± SD; *p < 0.01, **p < 0.001).

(C) In vitro translation was performed using luciferase-encoding mRNA containing A or 50% m⁶A and with or without a 5′cap as indicated. While unmethylated, capped mRNA + 10 pmole eIF4E is robustly translated, the unmethylated, uncapped mRNA fails to be translated. However, m⁶A-containing mRNA is efficiently translated even when no 5′ cap is present (n = 3; mean ± SD; *p < 0.01).

(D) m⁶A residues in the coding sequence do not induce cap-independent translation. Uncapped, luciferase-encoding mRNAs containing either the natural β-globin 5′ UTR or a modified β-globin 5′ UTR containing either zero, one, or three A residues as indicated were used for in vitro translation assays. Translation of m⁶A-containing mRNA with zero A residues in the 5′ UTR was markedly diminished, indicating that coding sequence m⁶A residues are unable to induce cap-independent translation. However, when a single m⁶A was added to the 5′ UTR, the transcripts were robustly translated. Methylated 5′ UTRs with a single A near the 5′ end, the middle (mid), or near the 3′ end all showed similar levels of translation (n = 3; mean ± SD; **p < 0.001, ***p < 0.0001, ****p < 0.00001). Schematic shows the distribution of A residues within each β-globin 5′ UTR variant (the unmodified β-globin 5′ UTR contains 17 A residues).

(E) mRNA with a single m⁶A within the 5′ UTR and no m⁶As in the remainder of the transcript induces cap-independent translation. Uncapped, luciferase-encoding mRNAs, which contained either a single adenosine 5′-monophosphate (AMP) or N⁶-methyladenosine 5′-monophosphate (m⁶AMP) at the 5′ end, were used for in vitro translation. Only the m⁶A-containing mRNA was translated, demonstrating that a single 5′ end m⁶A residue is capable of inducing cap-independent translation (n = 3; mean ± SD; **p < 0.001). The reduced translation efficiency of this mRNA compared to mRNAs with internally methylated 5′ UTRs is likely due to inefficient incorporation of m⁶A residues at the 5′ end by T7 RNA polymerase.

See also Figure S1.