Selective RNA sequestration in biomolecular condensates directs cell fate transitions

Recent studies have emphasized the significance of biomolecular condensates in modulating gene expression through RNA processing and translational control. However, the functional roles of RNA condensates in cell fate specification remains poorly understood. Here, we profiled the coding and non-coding transcriptome within intact biomolecular condensates, specifically P-bodies, in diverse developmental contexts, spanning multiple vertebrate species. Our analyses revealed the conserved, cell type-specific sequestration of untranslated RNAs encoding key cell fate regulators. Notably, P-body contents did not directly reflect active gene expression profiles for a given cell type, but rather were enriched for translationally repressed transcripts characteristic of the preceding developmental stage. Mechanistically, microRNAs (miRNAs) direct the selective sequestration of RNAs into P-bodies in a context-dependent manner, and perturbing AGO2 or alternative polyadenylation profoundly reshapes P-body RNA content. Building on these mechanistic insights, we demonstrate that modulating P-body assembly or miRNA activity dramatically enhances both activation of a totipotency transcriptional program in naïve pluripotent stem cells as well as the programming of primed human embryonic cells towards the germ cell lineage. Collectively, our findings establish a direct link between biomolecular condensates and cell fate decisions across vertebrate species and provide a novel framework for harnessing condensate biology to expand clinically relevant cell populations. For m2c cell sequencing, mES cells harboring 2C-specific reporters, Mervl-tdTomato and Zscan4-GFP were sorted based on reporter expression. Mervl/Zscan4 double-positive cells or negative cells with or without Ddx6 knockdown were sequenced. For hESC samples, a naive ES cell line carrying the totipotency specific reporter TPRX1-GFP was sorted based on reporter expression. TPRX1 positive cells and TPRX1 negative cells were sorted and sequenced in DDX6 KD or Ctrl KD conditions. This was repeated in primed conditions. For germ cell differentiations, human BTAG iPSCs harboring PGC specific reporters for TFAP2C-GFP and BLIMP1-TOMATO were used. TFAP2C/BLIMP1 double positive and negative cells were sorted and sequenced in DDX6 and Ctrl KD conditions, in naive conditions, and then after differentiation in iMELCs, and then PGCs.