ZFIN ID: ZDB-FIG-200121-7
Yang et al., 2019 - RNA 5-Methylcytosine Facilitates the Maternal-to-Zygotic Transition by Preventing Maternal mRNA Decay. Molecular Cell   75(6):1188-1202.e11 Full text @ Mol. Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagents:
Anatomical Term:
Stage Range: 64-cell to Shield
PHENOTYPE:
Fish:
Knockdown Reagents:
Observed In:
Stage Range: 64-cell to Bud

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.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
cap1 AB standard conditions 64-cell to Shield embryonic structure RTPCR
AB + MO1-pabpc1a standard conditions 64-cell to Shield embryonic structure RTPCR
AB + MO1-pabpc1a + MO3-ybx1 standard conditions 64-cell to Shield embryonic structure RTPCR
AB + MO3-ybx1 standard conditions 64-cell to Shield embryonic structure RTPCR
tex2 AB standard conditions 64-cell to Shield embryonic structure RTPCR
AB + MO1-pabpc1a standard conditions 64-cell to Shield embryonic structure RTPCR
AB + MO1-pabpc1a + MO3-ybx1 standard conditions 64-cell to Shield embryonic structure RTPCR
tpp2 AB standard conditions 64-cell to Shield embryonic structure RTPCR
AB + MO1-pabpc1a standard conditions 64-cell to Shield embryonic structure RTPCR
AB + MO1-pabpc1a + MO3-ybx1 standard conditions 64-cell to Shield embryonic structure RTPCR
AB + MO3-ybx1 standard conditions 64-cell to Shield embryonic structure RTPCR
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
AB + MO1-pabpc1a standard conditions 64-cell - Shield embryonic structure cap1 expression decreased amount, abnormal
64-cell - Shield embryonic structure tex2 expression decreased amount, abnormal
64-cell - Shield embryonic structure tpp2 expression decreased amount, abnormal
Shield gastrulation delayed, abnormal
Bud gastrulation increased duration, abnormal
Bud somitogenesis delayed, abnormal
AB + MO1-pabpc1a + MO3-ybx1 standard conditions 64-cell - Shield embryonic structure cap1 expression decreased amount, abnormal
64-cell - Shield embryonic structure tex2 expression decreased amount, abnormal
64-cell - Shield embryonic structure tpp2 expression amount, ameliorated
Shield gastrulation delayed, abnormal
Bud gastrulation increased duration, abnormal
Bud somitogenesis delayed, abnormal
AB + MO3-ybx1 standard conditions 64-cell - Shield embryonic structure cap1 expression decreased amount, abnormal
64-cell - Shield embryonic structure tpp2 expression decreased amount, abnormal
Shield gastrulation delayed, abnormal
Bud gastrulation increased duration, abnormal
Bud somitogenesis delayed, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Molecular Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Mol. Cell