During mammalian early embryonic development, epiblast stem cells (ECs) acquire pluripotency, the unique capacity to differentiate into all cell types of an organism. This ability is implemented by specific gene expression programs established through epigenetic, transcriptional, and post-transcriptional regulations. Today, many studies have emphasized the central role of translation in integrating upstream transcriptional and post-transcriptional cues to control Pluripotent Stem Cell (PSC) identity. Therefore, a better understanding of translational regulations in PSCs is essential for uncovering molecular processes underlying specific cellular identity and controlling cell fate transitions. Translation is ensured by ribosome, a micromachinery composed of both proteins and RNA (rRNAs). While ribosome composition has long been thought to be invariable, it now appears that ribosomes with different compositions exist, and that these may provide them specialized translational activities. rRNA modifications, such as 2'O-ribose methylation (2'Ome) are an emerging integral part of this heterogeneity. 2’Ome concerns 112 sites in human rRNA, which are specifically guided by small nucleolar RNAs (snoRNAs, or SNORD), often located in key functional sites of ribosomes. We and others contributed to identify 2’Ome heterogeneity in a panel of physiological and pathological contexts. However, whether variations of rRNA 2’Ome occur in PSCs and regulate PSC translation programs by modulating ribosome activity to support underlying cellular properties remain to be explored.

In this context, we applied RiboMeth-Seq (a recent high-throughput technique to analyze 2’Ome) in a panel of murine pluripotent and differentiated cells and identified rRNA sites that display differential 2’Ome upon differentiation. Importantly, few sites are located in expansion segments of ribosomes, which were recently shown to modulate Hox mRNA translation (Leppek et al., 2020). We found 28S_Am1137 site to be 2’O-methylated only in PSC, and met first clues suggesting a pluripotent-specific regulation of its snoRNA (SNORD126) and the subsequent methylation. For instance, we identified impaired polysomal population upon 28S_Am1137 2’Ome’s abolition and now aim to identify whether such variation is implicated in the pluripotent identity, notably through translational regulation.