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RNA structure plays a fundamental role in internal initiation of translation. Picornavirus internal ribosome entry site (IRES) are long, efficient cis-acting elements that recruit the ribosome to internal mRNA sites. However, little is known about long-range constraints determining the IRES RNA structure. Here, we sought to investigate the functional and structural relevance of the invariant apical stem of a picornavirus IRES. Mutation of this apical stem revealed better performance of G:C compared with C:G base pairs, demonstrating that the secondary structure solely is not sufficient for IRES function. In turn, mutations designed to disrupt the stem abolished IRES activity. Lack of tolerance to accept genetic variability in the apical stem was supported by the presence of coupled covariations within the adjacent stem-loops. SHAPE structural analysis, gel mobility-shift and microarrays-based RNA accessibility revealed that the apical stem contributes to maintain IRES RNA structure through the generation of distant interactions between two adjacent stem-loops. Our results demonstrate that a highly interactive structure constrained by distant interactions involving invariant G:C base pairs plays a key role in maintaining the RNA conformation necessary for IRES-mediated translation.

The foot-and-mouth disease virus (FMDV) RNA contains two in-frame AUG codons separated by 84 nt that direct translation initiation of the viral polyprotein. The mechanism of initiation at the IRES-proximal AUG codon (AUG1) has been previously analyzed, whereas no data on factor requirements for AUG2 have been reported. Here, using the method of 48S translation initiation complex reconstitution, we show that eIF1 is indispensable in forming the 48S initiation complex at AUG2. In contrast, it reduces the assembly of this complex at AUG1. Stabilization of a stem-loop between the initiation triplets induces a small decrease in the toeprint intensity at AUG2, accompanied by an increase in the AUG1/AUG2 ratio as well as a moderate reduction of protein synthesis initiated at AUG2 in transfected cells. PTB and ITAF45 exerted an additive positive effect on the 48S complex at AUG2, although a substantial reconstitution on both AUGs occurs on omission of either of these proteins. Relative to the beta-globin mRNA, the 48S complex formation at AUG1 and AUG2 is slow and occurs with the same kinetics as revealed by the "kinetic" toeprint assay. Mutation of AUG1 to AUA does not abrogate protein synthesis in transfected cells, and has no effect on the rate of the 48S complex formation at AUG2. We conclude that the AUG2 initiation region is selected independently of 48S complex formation at the upstream AUG1. The kinetic toeprint assay also shows that cap-dependent assembly of the 48S complex in vitro occurs faster than the FMDV IRES-mediated complex assembly.