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. 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基因表达在翻译层面的广泛调控，已成为蛋白质表达时空调控的关键节点。内部核糖体进入位点（Internal ribosomal entry sites, IRESes）是核糖体实现精细化基因调控的一类重要机制。然而，对其开展严谨的功能表征仍颇具挑战。本研究搭建了一套适用于胚胎与细胞的多技术通用工具箱，涵盖单分子mRNA异构体成像、PacBio长读长测序、基于多聚核糖体谱的异构体特异性mRNA定量分析，以及环状RNA（circular RNA, circRNA）报告基因的IRES样翻译检测体系，为解析IRES介导的基因调控机制提供了全新的研究路径。我们针对胚胎Hoxa mRNA中存在争议的IRES样RNA元件展开研究，明确了携带IRES样元件的Hoxa9 mRNA异构体在特定胚胎组织中的相对表达、定位与翻译情况，并验证了circRNA中依赖Hoxa IRES样元件的翻译过程。借此，我们为阐明IRES样元件在基因调控与胚胎发育中的作用提供了全新的技术资源。本研究从小鼠E11.5胚胎中显微分离体节与神经管，采用PacBio SMRTbell 3.0建库试剂盒制备Iso-Seq文库，随后通过Sequel II测序平台完成测序。