The Role of Secondary and Tertiary Structure in the Cap-Independent Translation of FGF-9 and HIF-1-alpha
Under normoxic conditions, eukaryotes initiate translation of RNA through eIF4E recognition of the 5’ cap. However, under cellular stress, eukaryotic translation must be initiated through a 4E-independent, or “cap-independent” mechanism, involving eukaryotic initiation factor 4G (eIF4G) binding directly to the 5’ untranslated regions (5’ UTR) of the RNA. eIF4G binding then recruits the ribosome to the transcript. While this mechanism is useful for translation of apoptotic transcripts and transcripts involved in cell survival, cap-independent translation is also utilized by oncogenic RNA for tumorigenesis. Previous work by our lab and others has categorized this recruitment and initiation mechanism as either internal-ribosome-entry-site (IRES)-like, where the ribosome is recruited at or near the RNA start codon, or cap-independent-translational-enhancer (CITE)-like, where the 5’ UTR must have an unpaired, accessible 5’ end for successful ribosomal recruitment. IRES-like and CITE-like transcripts bind the same eukaryotic initiation factors, such as the eIF4G homolog death-associated-protein-5 (DAP5); however, to date, no significant sequence similarity or conserved secondary structure has been proposed as DAP5 or eIF4G binding sites. RNA structure, however, has previously been found to bind initiation factors in viral IRES. Using selective 2’ hydroxyl acylation by primer extension (SHAPE), I found that the FGF-9 5’ UTR RNA, an oncogenic IRES-like translational enhancer, and the HIF-1-alpha 5’ UTR RNA, an oncogenic CITE-like translational enhancer, take on complex, distinct secondary structures. Protein toeprinting, DAP5 SHAPE footprinting, and UV crosslinking were used to map the binding sites of DAP5 along the FGF-9 and HIF-1-alpha 5’ UTR RNAs. Lastly, I used bioinformatics software to propose a three-dimensional binding model of DAP5 to these RNAs. DAP5 appears to bind specific, but distinct, surfaces on the structures of the FGF-9 and HIF-1-alpha 5’ UTR RNAs. I propose that protein-RNA interactions may involve more of these binding surfaces, rather than individual motifs. Higher-order structures may make attractive chemotherapeutic targets in and of themselves, or they may be attached ahead of RNA-based therapeutic sequences as dosing mechanisms.
Biochemistry, Biological and Chemical Physics, Biology, Biophysics, Cancer Biology, Computational Biology, Genetics, Genomics, Molecular Biology, Molecular Genetics, Other Genetics and Genomics, Structural Biology, RNA, FGF9, HIF1a, RNA structure, RNA translation, cap independent translation
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