On December 7, Zhu Xueliang's research group at Hangzhou Institute for Advanced Study, UCAS and the Center for Excellence in Molecular Cell Science, CAS, and Jing Naihe's research group at the Center for Excellence in Molecular Cell Science, CAS and Guangzhou Lab, jointly published a paper titled "Maternal mRNA deadenylation and allocation via Rbm14 condensates facilitates vertebrate blastula development" in the international academic journal EMBO Journal, discovering that the cytoplasmic condensates formed by the co-phase separation of Rbm14 with maternal m6A-mRNAs and deadenylase Parn exercise some of the deadenylation, sequestration and allocation functions of maternal mRNAs, thereby facilitating vertebrate blastula development.
The genome of fertilized eggs is initiallytranscriptionally silent, and maternal mRNAs accumulated from oocytes are gradually segregated intodaughter blastomeres as cleavage progresses, providing protein translation templates for early embryonic development. With Zygotic Genome Activation (ZGA), gene transcription becomes more vigorous and maternal mRNAs are cleared accordingly until embryos are completely dependent on genomically transcribed mRNAs. This process, known as the maternal-to-zygotic transition, varies widely across species. For example, the dependence of zebrafish embryos on maternal mRNAs lasts from the 1-cell phase to at least the 1000-cell phase, while the maternal mRNAs of mouse embryos degrade significantly after the 2-cell phase. The protein translation activity and stability of maternal mRNAs are finely regulated. In species such as fruit flies, zebrafish, and African clawed toads, some maternal mRNAs undergo deadenylation rapidly after egg fertilization, resulting in inhibition of translation activity, and then poly(A) tails are regained through cytoplasmic polyadenylation. Interestingly, mRNAs with deadenylation are usually degraded quickly, but they remain stable for several hours in early embryos, and the underlying mechanism is still unclear.
Interesting phenomena of nucleocytoplasmic localization changes
In previous studies, the research group found that nuclear RNA-binding protein Rbm14 formed condensates through the intrinsically disordered region (IDR) at the C-terminal and regulated the formation of the dorsal-ventral axis in the gastrula period of zebrafish.Since Rbm14 is also a maternal protein factor, the authors studied its localization in the early embryos of zebrafish and were surprised to find that Rbm14 was located in the cytoplasm and formed abundant condensates from the 4-cell phase (1 h post-fertilization) to ZGA (the 1000-cell phase, which is 3 h post-fertilization). The condensates gathered at spindle poles in the mitotic phase by associating with centrosomal g-tubulin puncta and displayed asymmetrical distribution across the embryonic midline in 8- and 16-cell embryos, which indicates that they could be unequally segregated into daughter blastomeres. The condensates gradually disappeared with the start of ZGA, and Rbm14 was gradually localized in the nucleus (Figure 1).
Figure 1. Localization of Rbm14 in the early embryonic development of zebrafish.A. Rbm14 gradually changed from cytoplasmic localization to nuclear localization during cleavage at 1-6 hours post-fertilization (hpf). Rbm14 is marked in green and DNA (DAPI) in purple. B.The Rbm14 condensates gathered at spindle poles in the mitotic phase and displayed asymmetrical or asymmetrical distribution. From left to right:8-cell embryos, 16-cell embryos and 64-cell embryos. Rbm14 is marked in green, g-Tubulin in blue and DNA (DAPI) in red.
Spatiotemporally regulated protein-RNA condensates
Does thenucleoprotein Rbm14 form condensates in the cytoplasm due to its binding to certain cytoplasmic factors? Considering the presence of a large amount of maternal mRNAs in the cytoplasm during the maternal control phase, the researchers first used the RNA dye for staining, and the results showed that Rbm14 condensates were rich in maternal RNAs. N6-methyladenosine (m6A) and 5-methylcytosine (m5C) modifications were reported in the maternal mRNAs of zebrafish. The level of m6A modification gradually decreased after reaching the maximum at 2 hpf, and the level of m5C modification gradually increased. In vitro phase separation experiments demonstrated thatnon-methylation modified RNA and m6A-RNA promoted the formation of the condensates and their separation with Rbm14, whereas m5C modification inhibited the formation of the condensates.
The researchers further explored the physiological function of Rbm14 condensates: Depletion of Rbm14 in both zebrafish and mice resulted in development arrest at the blastula stage and embryonic lethality, indicating the importance of Rbm14 for blastula development; they carried out Rbm14 knockdown and rescue experiments in zebrafish embryos, proving that phase separation and RNA binding of Rbm14 were essential for blastula development.The results of deep RNA sequencing showed that there were abundant maternal mRNAs accumulated in zebrafish embryos with Rbm14 depletion, and the in vitro poly(A) tail length determination experiment confirmed that the poly(A) tail of some maternal mRNAs was not subject to deadenylation. The researchers further found that the deadenylase Parn interacted with Rbm14 and promoted the formation of Rbm14 condensates through co-phase separation. Moreover, the deadenylase activity of Parn in the Rbm14 condensates was high.
According to the experimental results, the researchers proposed the molecular mechanism model of Rbm14 condensates regulating early embryonic development at the spatiotemporal level (Figure 2): The Rbm14 condensates facilitate deadenylation of maternal mRNAs through Parn and temporarily sequestrate the mRNAs to avoid degradation, and they are segregated into daughter blastomeres in a symmetrical or asymmetrical manner during the mitotic phase.As development progresses, the Parn level decreases, the m5C modification increases, and Rbm14 condensates gradually disperse and release mRNAs without poly(A) tail, which can be cleared by degradation or re-activated by polyadenylation. In this way, cell proliferation, differentiation and maternal-to-zygotic transition proceed normally, facilitating blastula-to-gastrula development.
Figure 2. Model of Rbm14 condensates regulating early embryonic development.
The study revealed that Rbm14 condensates dynamically and finely regulated the translational activity and stability of maternal mRNAs in vertebrates by coordinating processes such as deadenylation, polyadenylation, and m6A and m5C modification of the maternal mRNA.The researchers speculated that Rbm14 condensates may also regulate cell proliferation and fate by equally or differentially allocating maternal mRNAs to daughter blastomeres during the mitotic phase.
Xiao Yue, a post-doctor at the Hangzhou Institute for Advanced Study, UCAS, and Chen Jiehui, a doctoral student (now graduated) at the Center for Excellence in Molecular Cell Science, CAS are the co-first authors of the paper.The research was assisted by research fellow Li Jingsong, Dr. Yang Suming, Sun Honghua, and Xie Lele from the Center for Excellence in Molecular Cell Science, and supported by the National Key R&D Program, the Strategic Priority Research Program of the Chinese Academy of Sciences, the National Natural Science Foundation of China, and the Zhejiang Provincial Postdoctoral Science Foundation.
Link of the paper: https://www.embopress.org/doi/abs/10.15252/embj.2022111364
Source | School of Life Science
Editor | Chu Qinyu
Executive Editor | Wang Xia
