RNA–protein interaction methods to study viral IRES elements

• Functional analysis of viral IRES elements.
• Analysis of RNA-binding proteins interacting with viral IRES elements.
• Structural analysis of viral IRES elements by SHAPE and DMS probing.
• Approaches to study the impact of RNA-binding proteins on IRES RNA structure.

Translation control often takes place through the mRNA untranslated regions, involving direct interactions with RNA-binding proteins (RBPs). Internal ribosome entry site elements (IRESs) are cis-acting RNA regions that promote translation initiation using a cap-independent mechanism. A subset of positive-strand RNA viruses harbor IRESs as a strategy to ensure efficient viral protein synthesis. IRESs are organized in modular structural domains with a division of functions. However, viral IRESs vary in nucleotide sequence, secondary RNA structure, and transacting factor requirements. Therefore, in-depth studies are needed to understand how distinct types of viral IRESs perform their function. In this review we describe methods to isolate and identify RNA-binding proteins important for IRES activity, and to study the impact of RNA structure and RNA–protein interactions on IRES activity.