A versatile IRES toolbox to determine IRES-like activity exemplified by Hoxa mRNA expression and translation in mouse embryonic tissues
Widespread control of gene expression at the level of translation has emerged as a key point of protein expression regulation in space and time. Internal ribosomal entry sites (IRESes) are a prominent mechanism by which ribosomes can confer greater gene regulation. However, their rigorous functional characterization remains difficult. Here we present a versatile toolbox of technologies in embryos and cells including single-molecule mRNA isoform imaging, Pacbio long read sequencing, isoform-sensitive mRNA quantification along polysome profiles, and IRES-like translation of circular RNA (circRNA) reporters as a new guide to understanding IRES-mediated regulation. We investigate the disputed IRES-like RNA elements in embryonic Hoxa mRNAs. We show the relative expression, localization and translation of the IRES-like containing Hoxa9 mRNA isoform in specific embryonic tissues, and Hoxa IRES-like dependent translation in circRNAs. We thereby provide a new resource of technologies to elucidate the roles of IRES-like elements in gene regulation and embryonic development. Overall design: somite and neural tube was microdissected from mouse E11.5 embryo. Iso-seq library was prepared using PacBio SMRTbell 3.0 kit, and then sequenced using Sequel II