RNA-binding proteins with intrinsically disordered regions (IDRs) such as Rbm14 can phase separate in vitro. To what extent the phase separation contributes to their physiological functions is however unclear. Here we show that zebrafish Rbm14 regulates embryonic dorsoventral patterning through phase separation. Zebrafish rbm14 morphants displayed dorsalized phenotypes associated with attenuated BMP signaling. Consistently, depletion of mammalian Rbm14 downregulated BMP regulators and effectors Nanog, Smad4/5, and Id1/2, whereas overexpression of the BMP-related proteins in the morphants significantly restored the developmental defects. Importantly, the IDR of zebrafish Rbm14 demixed into liquid droplets in vitro despite poor sequence conservation with its mammalian counterpart. While its phase separation mutants or IDR failed to rescue the morphants, its chimeric proteins containing an IDR from divergent phase separation proteins were effective. Rbm14 complexed with proteins involved in RNA metabolism and phase separated into cellular ribonucleoprotein compartments. Consistently, RNA deep sequencing analysis on the morphant embryos revealed increased alternative splicing events as well as large-scale transcriptomic downregulations. Our results suggest that Rbm14 functions in ribonucleoprotein compartments through phase separation to modulate multiple aspects of RNA metabolism. Furthermore, IDRs conserve in phase separation ability but not primary sequence and can be functionally interchangeable.

Early embryonic development depends on proper utilization and clearance of maternal transcriptomes. How these processes are spatiotemporally regulated remains unclear. Here we show that nuclear RNA‐binding protein Rbm14 and maternal mRNAs co‐phase separate into cytoplasmic condensates to facilitate vertebrate blastula‐to‐gastrula development. In zebrafish, Rbm14 condensates were highly abundant in blastomeres and markedly reduced after prominent activation of zygotic transcription. They concentrated at spindle poles by associating with centrosomal γ‐tubulin puncta and displayed mainly asymmetric divisions with a global symmetry across embryonic midline in 8‐ and 16‐cell embryos. Their formation was dose‐dependently stimulated by m6A, but repressed by m5C modification of the maternal mRNA. Furthermore, deadenylase Parn co‐phase separated with these condensates, and this was required for deadenylation of the mRNAs in early blastomeres. Depletion of Rbm14 impaired embryonic cell differentiations and full activations of the zygotic genome in both zebrafish and mouse and resulted in developmental arrest at the blastula stage. Our results suggest that cytoplasmic Rbm14 condensate formation regulates early embryogenesis by facilitating deadenylation, protection, and mitotic allocation of m6A‐modified maternal mRNAs, and by releasing the poly(A)‐less transcripts upon regulated disassembly to allow their re‐polyadenylation and translation or clearance.

Background: To identify the aortic valve spatial location information through spatial transcriptomics (ST) technology and explore the spatial expression and role in valve calcification of the key factors at the transcriptome-wide scales, thus providing new insight for clinical prevention and treatment of calcific aortic valve disease (CAVD). Methods: Six cases of human active valve stenosis calcified valves from valve replacement surgery and two cases of relatively normal aortic valves from heart transplantation were collected between March 1, 2021 and September 1, 2021. We constructed tissue-wide gene expression sequencing during aortic valve calcification formation using ST and performed comprehensive mapping of differentiation transitions and spatial expression information in the calcified area of the valve. Results: We identified 6736 genes based on the count level of gene expression at different locations and obtained the spatial distribution map of the three-layer structure of the main 9 cells of the aortic valve during the formation of valve calcification and proportions of aortic valves. Shear stress factors were significantly enriched in the aortic valve. The differential expression of TRP family genes was significantly based on spatial transcriptome analysis. Conclusion: Herein, we elucidated the spatial resolution of the transcriptome and tissue scope of the adult aortic valve, providing a basis for a better understanding of the cellular origin and complex cytopathological differentiation process of CAVD. Our findings further the understanding of the pathogenesis and heterogeneity of aortic valve calcification and the implementation of more effective personalized treatment methods.