Hwang H et al. (2023), Phase transition of maternal RNAs during verteb...

XB-ART-59806

bioRxiv 2023 May 11; doi: 10.1101/2023.05.11.540463.

Show Gene links Show Anatomy links

Phase transition of maternal RNAs during vertebrate oocyte-to-embryo transition.

Hwang H , Chen S , Ma M , Divyanshi , Fan HC , Borwick E , Böke E , Mei W , Yang J .

???displayArticle.abstract???
The oocyte-to-embryo transition (OET) is regulated by maternal products stored in the oocyte cytoplasm, independent of transcription. How maternal products are precisely remodeled to dictate the OET remains an open question. In this work, we discover the dynamic phase transition of maternal RNAs during Xenopus OET. We have identified 863 maternal transcripts that transition from a soluble state to a detergent-insoluble one after oocyte maturation. These RNAs are enriched in the animal hemisphere and many of them encode key cell cycle regulators. In contrast, 165 transcripts, including nearly all Xenopus germline RNAs and some vegetally localized somatic RNAs, undergo an insoluble-to-soluble phase transition. This phenomenon is conserved in zebrafish. Our results demonstrate that the phase transition of germline RNAs influences their susceptibility to RNA degradation machinery and is mediated by the remodeling of germ plasm. This work thus uncovers novel remodeling mechanisms that act on RNAs to regulate vertebrate OET.

???displayArticle.pubmedLink??? 37214813
???displayArticle.pmcLink??? PMC10197690
???displayArticle.link??? bioRxiv

???attribute.lit??? ???displayArticles.show???

	Fig 2. Soluble-to-insoluble phase transition of RNA during oocyte maturation.A. MA plot shows the percentage of RNA in the pellet fraction in the mature egg (X-axis) and the ratio between the RNA in the pellet fraction of the egg and that of the oocyte (Y-axis). S-I transcripts are highlighted in the magenta box. B. Gene ontology analysis demonstrates the top biological processes that are enriched among S-I transcripts. C. Fractionation RT-qPCR was performed to validate fractionation RNA-seq results. The percentage distribution of ccna1, wee2.S, hmmr.L, parpbp.L, cep152.L, lig4.L, larp1b.S, exd3.L, pif1.L, sox3.S, sema3d.S, ssx2ip.L, espl1.L, wee2.L, ncapd2.S, fbxo43.L, cdc6.L, ccdc18.L, zbtb12.L, kank1.L, ccnb1.2.L, ncbp1.S, eif2ak3.S, dock7.S, ccnb2.L, dbr1.L, and rad21.L in the supernatant and pellet fractions were calculated. D. The percentage distribution of all markers analyzed in C was combined and plotted into the graph. E. Heat maps show the classification of maternal transcripts based on their degradation during the MZT. Class A transcripts are most rapidly degraded. Class B transcripts are degraded relatively slowly. Class C transcripts are relatively stable during early development. F. Venn diagram shows the majority of S-I transcripts belong to Class A. G. Heatmap shows the expression of S-I RNAs during early embryonic development.
	Fig 3. Insoluble-to-soluble phase transition of RNA during oocyte maturation.A. MA plot shows the percentage of RNA in the pellet fraction in the oocyte (X-axis) and the ratio between the RNA in the pellet fraction of the egg and that of the oocyte (Y-axis). I-S transcripts are highlighted in the red box. B. Gene ontology analysis demonstrates the top biological processes that are enriched among I-S transcripts. C. Protein-Protein Interaction (PPI) map shows the transcripts selected from a red box in panel A. This PPI shows only proteins interacting with at least one or more other proteins. Purple boxes indicate germline transcripts. D-G. Fractionation RT-qPCR was performed to validate fractionation RNA-seq results. D. The percentage distribution of germline I-S RNAs, including nanos1, xdazl, pgat, ddx25, grip2, sybu, dnd1, and xvelo1 in the supernatant and pellet fractions, were calculated. E. The percentage distribution of all germline RNAs analyzed in D was combined and plotted into the graph. F. The percentage distribution of somatic I-S RNAs, including vegT, gdf1, wnt11, elov11.S, wasl.S, and cdr2l.S in the supernatant and pellet fractions, were calculated. G. The percentage distribution of all somatic I-S RNAs analyzed in F was combined and plotted into the graph.
	Fig 4. Turnover of Xvelo1 during oocyte maturation results in the solubilization of germline RNAs.A. Xvelo1 protein is enriched in the insoluble fraction in the oocyte. Oocytes were lysed in NP-40 lysis buffer. After centrifugation, the lysate was separated into the supernatant (S) and pellet (P). Supernatant prepared from 10 oocytes was incubated with an anti-Xvelo1 antibody to enrich Xvelo1 in the soluble fraction. The supernatant, pellet, and IP samples were mixed with SDS sample buffer and subjected to western blotting. B. The expression of Xvelo1 in the oocytes, ovulated eggs, and embryos at stages 1, 6, 8.5, and 10 was analyzed by western blot. C. The expression of Xvelo1 during oocyte maturation was analyzed by IF. White arrowheads point to Xvelo1 remaining in the vegetal pole in mature eggs. The scale bars indicate 100 μm. D. Oocytes and mature eggs of the Dria transgenic frogs, which carry a mitochondria-specific GFP transgene, were stained with an anti-GFP antibody. The scale bars indicate 100 μm. E. Whole mount in situ results show nanos1 transcripts are located in punctate aggregates in the vegetal of the oocyte. After oocyte maturation, nanos1 transcripts show a diffuse appearance, with only a small number of puncta remaining in the vegetal pole. The scale bars indicate 200 μm. F and G. Overexpression of Xvelo1 prevents solubilization of germline RNAs after oocyte maturation. Oocytes were injected with 2 ng of Xvelo1 RNA, and cultured for 2 days, followed by progesterone treatment. Fractionation RT-qPCR was performed to assess the phase transition of nanos1, xdazl, pgat, ddx25, dnd1, sybu, and grip2 (F). G is the combination of all germline RNAs analyzed in F.
	Fig 5. Insoluble germline RNAs are resistant to RNase A in vitro.A. Crude oocyte lysate was treated with various doses of RNase A. After RNase A-treatment, RNA was extracted for RT-qPCR. The level of nanos1, ddx25, grip2, actin, hsc70, and h2a was measured. RNA from untreated lysate was set as 100%. B. Schematic diagram shows the procedure for the experiments in panel C. C. Crude oocyte lysate was treated with 12.5 pg/μl RNase A for various amounts of time. Degradation kinetics of germline (nanos1, xdazl, pgat, ddx25, grip2, sybu, dnd1, and xvelo1) and somatic (psma1, odc, gapdh, h2a, actin, hsc70, psme3, and ccna1) RNAs were measured by RT-qPCR. The expression of each germline and somatic RNAs in the oocyte and the mature egg was shown individually. The panel on the right side is the combination of all germline and somatic RNAs in the oocyte and egg. D. Schematic diagram shows the procedure of experiments in panel E. E. Crude lysate was treated with RNase A, separated into the soluble and insoluble fractions, followed by RT-qPCR for nanos1, xdazl, pgat, ddx25, grip2, sybu, dnd1, and xvelo1.
	Fig 6. Buc regulates the solubility and stability of germline RNAs in zebrafish.A. Wild type and bucky ball oocytes were stained with ER-tracker and Mito-tracker. Scale bars on the images for ER trackers indicate 20 μm, and those for Mito trackers indicate 10 μm. B. Fractionation RT-qPCR was performed to measure the solubility of nanos3, dnd, dazl, ca15b, gra, and vasa in fully-grown oocytes from the wild-type fish and bucky ball mutants. The right panel is the combination of all these germline RNAs. C. The expression of nanos3, dnd, dazl, ca15b, gra, and vasa in 1-cell stage embryos derived from the wild-type and bucky ball mutant females were assessed by RT-qPCR. Student’s t-tests were performed. p<0.01, **p<0.0001.
	Fig 7. RNA phase transition during zebrafish OET.Fractionation RT-qPCR was performed to measure the percentage distribution of ccna1, ccnb1, wee2, dazl, nanos3, gra, ca15b, dnd, and vasa in stage I, stage II/III, and fully-grown oocytes, ovulated eggs, and embryos at 2-, 32-, and 512-cell stages.

References [+] :

Aguero, Maternal Dead-end 1 promotes translation of nanos1 by binding the eIF3 complex. 2017, Pubmed, Xenbase