Kostiuk V et al. (2025), Nup107 contributes to the maternal-to-zygotic t...

XB-ART-61210

Development 2025 Feb 15;1522:. doi: 10.1242/dev.202865.

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Nup107 contributes to the maternal-to-zygotic transition by preventing the premature nuclear export of pri-miR427.

Kostiuk V , Kabir R , Levangie K , Empke S , Morgan K , Owens NDL , Lusk CP , Khokha MK .

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Emerging evidence suggests that the nuclear pore complex can have unique compositions and distinct nucleoporin functions in different cells. Here, we show that Nup107, a key component of the NPC scaffold, varies in expression over development: it is expressed at higher levels in the blastula compared to the gastrula, suggesting a crucial role before gastrulation in Xenopus. We find that depletion of Nup107 affects the differentiation of the early germ layers leading to an expansion of the ectoderm at the expense of endoderm and mesoderm. By analyzing an RNA-sequencing time course, we observed that depletion of Nup107 affects the maternal-zygotic transition by delaying the degradation of maternal transcripts that occurs as zygotic transcription begins. The transcripts are enriched in recognition sites for miR427, a conserved microRNA that destabilizes maternal transcripts including REST, which encodes a Kruppel-type zinc-finger transcription factor that we demonstrate is crucial for ectodermal cell fates. Mechanistically, we show that Nup107 is required to prevent the premature export of pri-miR427 transcript before processing. Nup107 depletion leads to the reduced production of mature miR427 and maternal transcript stabilization. We conclude that high levels of Nup107 in the early embryo are crucial for the nuclear retention and subsequent processing of pri-miR427 transcripts that is required for timely maternal RNA clearance to enable gastrulation.

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	Fig. 1. Spatio-temporal patterns of nucleoporin expression. (A) mRNA expression domains of nup107, nup62, and nup188 detected by whole-mount in situ hybridization using antisense probes at different developmental stages (panel above). Scale bar: 500 μm. (B) Protein expression levels of Nup62, Nup188, and Nup107 detected by western blotting at different developmental stages (panel above). Gapdh is a loading control. Three biological replicates were used for all experiments, with 30 embryos used per stage per condition. All data represent results from experiments replicated at least three times in the laboratory.
	Fig. 2. Nup107 depletion alters germ layer specification. (A) Representative images of whole-mount in situ hybridization embryos at stage 10 demonstrating reduced expression levels of dorsal mesodermal markers (chordin, goosecoid, foxj1) and upregulated expression of a ventral mesodermal marker (vent1) due to Nup107 depletion. (B) Whole-mount in situ hybridization images of embryos at stage 10 showing upregulated ectodermal marker (ectodermin) and reduced mesodermal marker (xbra) expression in Nup107-depleted embryos. Three biological replicates were used for all experiments. All data represent results from experiments replicated at least three times in the laboratory. Statistical significance was determined using two-way ANOVA analysis. *P<0.001, **P<0.0001. ns, not significant. Data are mean±s.d. Scale bars: 500 μm.
	Fig. 3. Depletion of Nup107 alters temporal differential expression of specific genes. (A,B) Summary of gene clusters with increased expression by heatmap (A) and cluster average (B). Clustered data is maximum normalized Gaussian process median expression for each gene. B provides mean±s.d. for each cluster. Decreased genes and ribozero clusters given in Fig. S7A,B. (C) Gene expression examples for each increased expression cluster for UIC and MO. Data points are gene expression in TPM; central line and shaded region are transformed Gaussian process median and 95% CI, respectively. (D) Enrichment of exact instances of microRNA seeds within 3′ UTRs of genes of each cluster. Color gives Fisher’s exact test −log10 P-value between the given number of instances and a gene being present in a cluster. Seeds organized by number of mismatches to miR427 sequences; see Materials and Methods for details. Schematic at top shows illustrates the experimental workflow. MO, nup107 MO; UIC, uninjected control.
	Fig. 4. miR427 expression levels at stage 8. (A) Northern blot showing relative levels of miR427 in comparison to total RNA amounts; miR427 OE and miR427 OE-P are positive overexpression controls. (B) qPCR quantification of miR427 expression levels using LNA probes for miR427 and u6 (small RNA standardization control). Three biological replicates were used for all experiments. All data represent results from experiments replicated at least three times in the laboratory. Statistical significance was determined using two-way ANOVA analysis. P<0.05, *P<0.01. ns, not significant. Data are mean±s.d.
	Fig. 5. miR427 overexpression rescues germ layer expression patterns associated with nup107 depletion. Increased ectodermal marker (ectodermin and foxi1a) expression due to Nup107 depletion (nup107 MO) was rescued by miR427 overexpression (nup107 MO+miR427). The overexpression of miR427 also rescued the reduced mesodermal signal (xbra) in Nup107-depleted embryos. Three biological replicates were used for all experiments. All data represent results from experiments replicated at least three times in the laboratory. Statistical significance was determined using two-way ANOVA analysis. P<0.01, **P<0.0001. ns, not significant. Scale bar: 500 μm.
	Fig. 6. rest is a potential target of miR427 and a regulator of ectodermal patterning. (A) Whole-mount in situ embryos at stage 8 demonstrating the effect of Nup107 depletion and miR427 overexpression on the changes in rest expression patterns. (B) Whole-mount in situ embryos at stage 10 demonstrating ectodermal marker (ectodermin and foxi1a) expression changes in Nup107- and Rest-depleted embryos. Three biological replicates were used for all experiments. All data represent results from experiments replicated at least three times in the laboratory. Statistical significance was determined using two-way ANOVA analysis. ****P<0.0001. ns, not significant. Scale bars: 500 μm.
	Fig. 7. The expression and subcellular localization of pri-miR427. (A) Whole-mount in situ hybridization demonstrating the expression domain of pri-miR427. Higher magnification images at lower right reveal nuclear staining (red arrowheads). Cell diagram was created in BioRender by Kostiuk, V., 2025. https://BioRender.com/x94m509. This diagram was sublicensed under CC-BY 4.0 terms, and subsequently annotated. (B) (Left) Using RNAScope, subcellular localization of the pri-miR427 (AS, antisense; S, sense) and odc1 transcript (a housekeeping gene control). (Right) Signal quantification of odc1 (top) and pri-miR427 (bottom). The following number of embryos were used for this experiment: sense pri-miR427 probe: 16 Nup107-depleted embryos and 20 control embryos; antisense pri-miR427 probe: 20 Nup107-depleted embryos and 18 control embryos; antisense odc1 probe: 19 Nup107-depleted embryos and 17 control embryos. The confidence intervals for measurements were: sense pri-miR427 probe: nup107 MO sample 1.3±0.6 (0.7-1.8), UIC sample 0.9±0.4 (0.6-1.3); antisense pri-miR427 probe: nup107 MO sample 10.3±3.4 (6.9-13.8), UIC sample 18.8±7.4 (11.4-26.2); antisense odc1 probe: UIC sample 5.7±0.8 (4.9-6.5), nup107 MO sample 6.3±0.8 (5.5-7.0). Three biological replicates were used for all experiments. All data represent results from experiments replicated at least three times in the laboratory. Statistical significance was determined using two-way ANOVA analysis. P<0.05 was considered statistically significant. ****P<0.0001. ns, not significant. Scale bars: 500 μm (A); 10 μm (B).