Fig. 5

Ybx1 and Pabpc1a Mediated m⁵C Protects Maternal mRNA Stability in Early Zebrafish Embryos

(A) Pabpc1a morphants display developmental arrest similar to that of ybx1 morphants or ybx1 and pabpc1a MO-coinjected embryos.

(B) qRT-PCR analysis showing the decreased expression of three representative genes in pabpc1a morphants or in embryos coinjected with ybx1 and pabpc1a MOs. Error bars represent mean ± SD; n = 3. ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, two-tailed Student’s t test.

(C) The in vivo degradation of cap1-WT or tpp2-WT mRNAs was faster in ybx1 morphants than in control embryos. mRNA abundance was determined by qRT-PCR. Error bars represent mean ± SD; n = 3. ^∗∗∗p < 0.001, two-tailed Student’s t test.

(D and E) Representative images (D) and quantification (E) of the relative expression levels of the WT products of cap1 and tpp2 in embryos with or without ybx1 deficiency at the sphere stage of development. The cap1-WT and tpp2-WT GFP reporter mRNAs produced lower GFP protein levels in ybx1 morphants than in control embryos. Scale bar, 500 μm. Error bars represent mean ± SD; n = 14. ^∗∗∗∗p < 0.0001, two-tailed Student’s t test.

(F) qRT-PCR showing the expression of cap1-WT in control embryos and ybx1 morphants at the indicated time points during MZT. RNA abundance was determined by qRT-PCR and normalized to the 0 hpf values. β-Actin served as an internal RNA control. Error bars represent mean ± SD; n = 3. ^∗p < 0.05, ^∗∗p < 0.01, two-tailed Student’s t test.

(G) qRT-PCR showing the expression of cap1- m⁵C (cyan) and cap1-C (black) reporter RNAs in control embryos at the indicated time points during MZT. RNA abundance was determined by qRT-PCR and normalized to 0 hpf values. β-Actin served as an internal RNA control. Error bars represent mean ± SD; n = 3. ^∗∗p < 0.01, ^∗∗∗p < 0.001, two-tailed Student’s t test.

(H) Schematic model of the dynamic regulation of maternal mRNA decay by m⁵C during zebrafish MZT. Maternal mRNAs without m⁵C modification are degraded rapidly through deadenylation during MZT, whereas a subset of maternal mRNAs with m⁵C modifications can be protected from degradation through m⁵C site recognition by the Ybx1 complex, which leads to the stabilization of mRNAs and subsequently enables the performance of their associated functions during early embryogenesis.

The p values in (B), (C), and (E)–(G) were all determined using two-tailed Student’s t tests.