Rbm24a interacts with germ plasm mRNAs and maintains their stability.The category of reads identified by RIP-seq. (B) Scatter plot showing the interaction between Rbm24a and a panel of mRNAs, with red dots highlighting germ plasm mRNAs. (C) GO analysis of Rbm24a-interacting transcripts. The hypergeometric test is employed for statistical assessment, and the Benjamini–Hochberg (BH) method is utilized for P value adjustment. (D) RIP-qPCR verification of Rbm24a binding to nanos3 mRNA, showing a more than 200-fold enrichment (n = 3 independent biological samples). Data are presented as mean ± SD. ****P < 0.0001, unpaired Student’s t test. (E) Localization analysis of nanos3 mRNA, Rbm24a-myc and Buc-GFP in HEK293 cells. Note that the simultaneous presence of Buc and Rbm24a efficiently recruits nanos3 mRNA into phase-separated granules. Scale bars, 10 μm. (F) Coexpression of nanos3 mRNA with Rbm24a, Buc, or both leads to an enhanced level of nanos3 transcripts (n = 3 independent biological samples). Data are presented as mean ± SD. **P < 0.01, ***P < 0.001 and ****P < 0.0001, unpaired Student’s t test. (G), RIP-qPCR in HEK293 cells showing the synergistic effect of Rbm24a and Buc in binding to nanos3 mRNA (n = 3 independent biological samples). Data are presented as mean ± SD. ****P < 0.0001, unpaired Student’s t test. (H) Temporally regulated degradation of maternal Rbm24a-GFP by zGrad mRNA injection. (I) Confocal imaging showing the progressive elimination of Rbm24a-GFP from the 64-cell stage onward. Piwil1 is also absent in zGrad-expressing embryos at 24 hpf, due to the absence of PGCs. Scale bars, 20 μm. (J) Degradation of Rbm24a in homozygous rbm24a-gfp KI embryos does not affect the formation of large germ plasm condensates at the 4-cell stage, but causes the absence of germ plasm mRNAs and PGCs in embryos at 6 hpf and 24 hpf. Scale bars, 200 μm. (K) FISH and IF experiments show the gradual disappearance of nanos3 mRNA and Piwil1 protein following zGrad mRNA injection. Notably, the size of nanos3- and Piwil1-positive germ granules is remarkably decreased at the 64-cell stage, and germ granules completely disappear by 24 hpf. Scale bars, 20 μm. (L) Working model of maternal Rbm24a function in germ plasm granule assembly. Rbm24a complexed with Buc serves as a reader to recruit and maintain germ plasm mRNAs in the condensates, promoting the formation of large germ plasm aggregates and protecting the stability of germ plasm mRNAs for differentiation of PGCs. Source data are available online for this figure.
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