P-bodies dynamics of hESCs by unveiling P-bodies properties of hESC and hESC-derived mesodermal cells
Processing bodies (P-bodies) are the membraneless organelles that play a critical role in RNA storage and degradation. P-body abnormalities contribute to diseases and developmental disorders, however, the dynamics of RNA components of P-bodies in human embryonic stem cells during maintenance and differentiation remain elusive. Here, we captured P-bodies during human embryonic stem cells(hESCs) to mesodermal differentiation using flow cytometry, and revealing unique transcriptomic features to other organelles and characterized by lower GC content and reduced M6A modifications. Genes enriched in P-body are cell-specific, mainly associated with translation regulation and the cytoskeleton, long non-coding RNAs in P-body tend to interact with RNAs, whereas cytoplasmic genes are conserved and essential for fundamental processes, and cytoplasmic lncRNAs mostly interact with proteins. During hESC to mesodermal differentiation, both the number of P-bodies and enriched genes were significantly reduced. In addition, P-bodies selectively attracted specific transposable elements in hESCs. Perturbation of P-body gene expression or overexpression of P-body core protein LSM14A could disrupt hESC differentiation but not maintenance. In conclusion, our data elucidate the dynamics of P-body during the differentiation of hESCs to the mesodermal lineage and support the link between P-body and stem cell pluripotency. Overall design: To investigate the properties of P-bodies in different cell types, we established tetON-LSM14A-GFP (P-bodies labelling) 293T and hESCs cell lines and purified P-bodies properties from 293T, hESCs, and hESC-derived mesodermal cells by fluorescence-activated cell sorting. To investigate the linkage of P-body and hESCs differentiation, we treat hESCs with 1µg/ml TGFßi, and analysis the gene expression profiles about the pluripotency markers. we treat tetON-LSM14A-GFP hESCs and derived mesodermal cells with or without 1µg/ml Doxycycline, and analysis the gene expression profiles of P-body core protein LSM14A overexpression in hESCs and hESC-derived mesodermal cells. After candidate screen, we construct CRISPRi-SPTAN1 knock-down hESCs, and comparatively analysis the gene expression of SPTAN1-KD in hESCs and hESC-derived mesodermal cells.