Abstract
Cap-independent translation initiation in eukaryotes involves initiation factor (eIF) binding to a transcript's 5' untranslated region (UTR). Internal-ribosome-entry-site (IRES)-like cap-independent translation initiation does not require a free 5' end for eIF binding, as eIFs recruit the ribosome to or near the start codon. For viral mRNA, recruitment usually utilizes RNA structure, such as a pseudoknot. However, for cellular mRNA cap-independent translation, no consensus RNA structures or sequences have yet been identified for eIF binding. Fibroblast-growth factor 9 (FGF-9) is a member of a subset of mRNA that are cap-independently upregulated in breast and colorectal cancer cells using this IRES-like method. Death-associated factor 5 (DAP5), an eIF4GI homolog, binds directly to the FGF-9 5' UTR to initiate translation. However, the DAP5 binding site within the FGF-9 5' UTR is unknown. Moreover, DAP5 binds to other, dissimilar 5' UTRs, some of which need a free 5' end to stimulate cap-independent translation. We propose that a particular RNA structure involving tertiary folding, rather than a conserved sequence or secondary structure, acts as a DAP5 binding site. Using SHAPE-seq, we modeled the FGF-9 5' UTR RNA's complex secondary and tertiary structure in vitro. Further, DAP5 footprinting and toeprinting experiments show DAP5's preference for one face of this structure. DAP5 binding appears to stabilize a higher-energy RNA fold that frees the 5' end to solvent and brings the start codon close to the recruited ribosome. Our findings offer a fresh perspective in the hunt for cap-independent translational enhancers. Structural, rather than sequence-specific, eIF binding sites may act as attractive chemotherapeutic targets or as dosage tools for mRNA-based therapies.