Comprehensive mutational analysis of the sequence–function relationship within a viral internal ribosome entry site

The cricket paralysis virus (CrPV) intergenic region internal ribosome entry site (IRES) binds to the ribosome without the need for any initiation factors. Their length, simple mechanism, and ability to function in diverse cell-free systems make CrPV-like IRESs useful tools to study the mechanism of translation and to express proteins. We report the use of a RelE-based next-generation sequencing method, termed SMARTI (sequencing-based mutational analysis of RNA translation initiation), to quantitatively determine the function of over 81 000 single and double mutants of CrPV IRES. The result is a comprehensive mutational database that serves as a consensus sequence-like analysis of IRES function. We have given particular attention to the sequence requirements within the three pseudoknots of the IRES element. The data indicate that each pseudoknot contains positions that are modifiable and mutation may even enhance IRES function through pseudotranslocation. CrPV IRES must balance being stable and dynamic as it forms the structure and ribosomal contacts required for translation initiation. Helical regions, especially in the transfer RNA-mimicking domain, are areas where flexibility may be especially beneficial. Moreover, we demonstrated that this high-throughput method is compatible with eukaryotic extract, providing an avenue for studying diverse eukaryotic RNA elements and for engineering sequences for protein expression.