RNA sequestration in cytoplasmic condensates regulates cell identity (ncRNA-Seq)
Post-transcriptional regulation is increasingly recognized as pivotal in guiding the gene expression programs that instruct cell fate decisions. Recent studies have highlighted the significance of RNA condensates in modulating gene expression through RNA processing and translational control. Yet, the functional roles and composition of RNA condensates in the context of cell fate specification remain unclear. In this study, we leveraged Fluorescence-Activated Particle Sorting to profile the coding and non-coding transcriptome within cytoplasmic condensates known as P-bodies across diverse developmental contexts and vertebrate species. Our analyses revealed the highly conserved, cell type-specific sequestration of fate-instructive RNAs, which are translationally suppressed and stored within P-bodies. Using degron systems in both human and mouse pluripotent cells, we demonstrate that the acute dissolution of P-bodies facilitates the translation of mRNAs otherwise suppressed in condensates, including genes associated with zygotic genome activation and totipotency. Consistently, loss of RNA sequestration awakens the totipotency transcriptional program in human and mouse pluripotent cells. Mechanistically, we provide evidence that miRNAs play a pivotal role in orchestrating the sequestration of specific RNAs into P-bodies in a context-dependent manner. Accordingly, perturbing AGO2, alternative polyadenylation, and miRNA function profoundly reshapes the sequestration of RNAs into P-bodies and alters cell fate. We further identify mir-300/381 as a key repressor of the totipotent transcriptional state. Collectively, our findings connect RNA processing within condensates to cell fate decisions across vertebrate species. Overall design: The hESC and mESC were differentiated into each of the cell types below, and then pbody contents were sorted and sequence, and cytoplasmic fractions were sequenced seperately. Two replicates were sequenced for each sample. In treatment or knockout conditions, each treatment condition was sequenced alongside a control cell line, two replicates of each for pbody and cytoplasm.