Distribution and structural diversity of Type IV internal ribosome entry sites

Internal Ribosome Entry Sites (IRESs) are RNAs that facilitate cap– and end-independent translation initiation in eukaryotes. Type IV IRESs, which include the hepatitis C virus IRES, directly bind the 40S subunit and require only a subset of the canonical initiation factors to function. As the full diversity and species distribution of these IRESs was unknown, we sought to identify and classify their full architectural variation. Using a secondary structure homology-based search method, we identified 163 putative Type IV IRESs from viruses with diverse hosts and phylogeny, including the first example in a double stranded viral genome. Clustering analysis based on the presence and overall size of secondary structure elements yielded three distinct groups, differentiated by secondary structure expansions and deletions. Chemical probing of representative IRESs from each cluster validated the predicted secondary structures and in vitro translation assays showed that structural differences correlate with functional variation. Our findings reveal distinct structural adaptations and patterns within the Type IV IRESs that may influence IRES function and mechanism.