Figure 1. 5′UTR m⁶A Enables Ribosome Binding to mRNA in the Absence of Cap-Binding Proteins.

(A) 5′ UTR methylation permits 48S initiation complex formation in the absence of the group 4 eIFs. In vitro transcribed, capped mRNAs encoding a MVHC tetrapeptide and containing either A or m⁶A were incubated with purified mammalian translation initiation components. Subsequent toeprinting analysis using a radiolabeled primer then revealed whether 48S initiation complexes were formed. Positions of the initiation codon, full-length cDNA, and the 48S complex are shown on the sides of the panel. Lanes C/T/A/G depict the corresponding DNA sequence. When unmethylated mRNA is used (lanes 1–5), 48S complexes are only formed when the cap-binding complex eIF4F is present (lanes 2 and 3). When eIF4F is absent, 48S complex formation on unmethylated mRNA is impaired (lanes 4 and 5). However, when mRNA with m⁶A in the 5′ UTR is used, 48S complex formation is observed even in the absence of eIF4F (lanes 9 and 10; compare to lanes 7 and 8 where eIF4F is present).

(B) eIFs1, 1A, and 3 are required for efficient m⁶A-induced cap-independent 48S complex formation. Toeprinting assays were performed as in (A) using A- or m⁶A-containing mRNAs and in the presence of various translation initiation components as indicated. m⁶A-containing mRNA exhibits robust 48S complex assembly in the absence of eIF4F, whereas A-containing mRNA does not (compare lanes 1 and 7). Efficient m⁶A-mediated 48S complex assembly is also dependent on the presence of eIFs1 and 1A, which is consistent with the known roles of these proteins in promoting scanning and AUG recognition (compare lanes 1 with lanes 2, 4, and 5). Removal of eIF3 also abolishes 48S complex assembly on m⁶A-containing mRNA (compare lanes 1 and 2), indicating that eIF3 is required for m⁶A-mediated 48S complex formation. Addition of 60S subunits, eIF5, eIF5B, eEF1H, eEF2, and aa-tRNAs resulted in the appearance of toeprints corresponding to pre-termination complexes at the stop codon, indicating that m⁶A-recruited 48S complexes are fully functional (lane 6).

(C) Omission of eIF2 from toeprinting assays results in the absence of 48S complexes (compare lanes 2 and 3), indicating that eIF2 is required for 48S complex assembly on m⁶A-containing mRNA.