A versatile toolbox to determine IRES activity in cells and embryonic tissues

Widespread control of gene expression through translation has emerged as a key level of spatiotemporal protein expression regulation. Internal ribosomal entry sites (IRESes) provide a prominent mechanism by which ribosomes can confer greater gene regulation. However, their rigorous functional characterization remains difficult. 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 guideline to understand IRES regulation. We investigate a broad range of cellular IRES RNA elements. We show IRES-dependent translation in circRNAs and 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.