47 High-resolution ribosome profiling reveals translational selectivity in the mammalian blastocyst

Abstract

Transcriptomic analyses of early mammalian embryos from multiple species have been comprehensively conducted in the last decade. However, mRNAs detected from overall transcriptomic profile of an embryo or a single cell do not necessarily represent their functional status, as there is a gap between the overall transcriptome and mRNAs that are actively translated. Ribosome profiling has been developed to infer the translational status of a specific mRNA species and thus analyse the genome-wide translatome. However, the broad application of ribosome profiling has been slowed by its complexity and the difficulty of adapting it to low-input samples such as embryos. In this study, we developed an ultra-low-input ribosome profiling protocol optimized for mammalian embryos and systematically analysed both polysome- and non-polysome-bound mRNA profiles of in vitro-produced bovine blastocysts. Ten equal fractions were collected by means of sucrose density gradient and ultracentrifugation of lysates from 100 pooled blastocysts (n = 2 pools), and subjected to RNA isolation and RNA sequencing. Our bioinformatics analyses of the mRNA profiles from each fraction along with the whole-transcriptome data revealed that compared with the overall transcriptome, there is a strong selection of mRNAs in the ribosome- and polysome-associated fractions, including transcriptional factors (e.g. POU5F1, ESRRB, AQP3, and APOA1) and genes involved in ribosome biogenesis, oxidative phosphorylation, and metabolic pathways, many of which are essential for the function of embryo implantation. We also identified novel epigenetic regulators selectively translated, including regulatory enzymes on histone modifications and RNA modifications (e.g. JMJD7, ALKBH4, ALKBH7, and METTL26). In addition, we confirmed the translation of the highly expressed, yet developmentally essential pathways in the blastocysts (e.g. Wnt and Notch signalling pathways). The selectively translated mRNAs were further validated by immunofluorescent staining at the protein level and cross-validated in both bovine and mouse blastocysts. Some of these genes show only modest expression in the overall transcriptome data. Their selective translation at the blastocyst stage is only being revealed by the ribosome fractions analyses, and their functions warrant future detailed investigations. In conclusion, this study reveals bona fide active translating mRNAs in the mammalian blastocyst. The low-input ribosome profiling protocol and the data presented here set an example and open future avenues for detailed ribosome fraction–based translatome analyses to reveal novel cellular/embryonic functional regulators beyond transcriptomic data.