A versatile toolbox for determining IRES activity in cells and embryonic tissues.

Widespread control of gene expression through translation has emerged as a key level of spatiotemporal regulation of protein expression. A prominent mechanism by which ribosomes can confer gene regulation is via internal ribosomal entry sites (IRESes), whose functions have however, remained difficult to rigorously characterize. Here we present a set of technologies in embryos and cells, including IRES-mediated translation of circular RNA (circRNA) reporters, single-molecule messenger (m)RNA isoform imaging, PacBio long-read sequencing, and isoform-sensitive mRNA quantification along polysome profiles as a new toolbox for understanding IRES regulation. Using these techniques, we investigate a broad range of cellular IRES RNA elements including Hox IRESes. We show IRES-dependent translation in circRNAs, as well as the relative expression, localization, and translation of an IRES-containing mRNA isoform in specific embryonic tissues. We thereby provide a new resource of technologies to elucidate the roles of versatile IRES elements in gene regulation and embryonic development.