Postnatal liver functional maturation requires Cnot complex-mediated decay of mRNAs encoding cell cycle and immature liver genes

ABSTRACT Liver development involves dramatic gene expression changes mediated by transcriptional and post-transcriptional control. Here, we show that the Cnot deadenylase complex plays a crucial role in liver functional maturation. The Cnot3 gene encodes an essential subunit of the Cnot complex. Mice lacking Cnot3 in liver have reduced body and liver masses, and they display anemia and severe liver damage. Histological analyses indicate that Cnot3-deficient (Cnot3−/−) hepatocytes are irregular in size and morphology, resulting in formation of abnormal sinusoids. We observe hepatocyte death, increased abundance of mitotic and mononucleate hepatocytes, and inflammation. Cnot3−/− livers show increased expression of immune response-related, cell cycle-regulating and immature liver genes, while many genes relevant to liver functions, such as oxidation-reduction, lipid metabolism and mitochondrial function, decrease, indicating impaired liver functional maturation. Highly expressed mRNAs possess elongated poly(A) tails and are stabilized in Cnot3−/− livers, concomitant with an increase of the proteins they encode. In contrast, transcription of liver function-related mRNAs was lower in Cnot3−/− livers. We detect efficient suppression of Cnot3 protein postnatally, demonstrating the crucial contribution of mRNA decay to postnatal liver functional maturation.

Nuclei are stained with DAPI (bottom panels). Top panels show merged images. Normal rabbit and mouse IgGs are used as negative controls.
Nuclei are stained with DAPI. Top panels in each genotype show merged images.
Magnified views of representative areas (inset). Scale bars, 100 μm. p value (log10) Figure S9. The upregulated mRNAs in Cnot3 -/livers are expressed at higher levels at 1 week of age than at 4 week age in wild-type livers.

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(A) Overlap of mRNAs upregulated in 4-week-old Cnot3 -/livers and those which decrease in wild-type livers from 1 to 4 weeks of age. Venn diagram of mRNAs that are expressed more than 2-fold higher in Cnot3 -/livers compared with controls at 4 weeks of age (red) and those that are expressed more than 3-fold higher in 1-week-old control livers compared with 4-week-old control livers (blue). Gapdhnormalized values are compared between 1-week-old and 4-week-old wild-type livers. Control hepatocytes Control 0h Control 6h Control 12h   Development: doi:10.1242/dev.168146: Supplementary information Development • Supplementary information Table S1. GO analysis of differently expressing mRNAs in 1w Cnot3 -/livers.

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Enriched GO terms of mRNAs upregulated (1) or downregulated (2) more than 2-fold in 1w Cnot3 -/livers compared to controls in microarray analysis were analyzed using DAVID. P-values, false discovery rates (FDRs) and gene lists included in GO terms are summarized. Please see also Supplementary figure 7A, C, D. Table S2. GO analysis of differently expressing mRNAs in 4w Cnot3 -/livers.
Enriched GO terms of mRNAs upregulated (1) or downregulated (2) more than 2-fold in 4w Cnot3 -/livers in microarray analysis compared to controls were analyzed using DAVID. P-values, FDRs and gene lists included in GO terms are summarized. Please see also Figure 4A, C, D. Table S3. GO analysis of mRNAs which show both maturation-dependent downregulation in control livers and upregulation in 4w Cnot3 -/livers.
Enriched GO terms of mRNAs which show both more than 3-fold decrease from 1 to 4w in control livers and more than 2-fold upregulation in 4w Cnot3 -/livers were analyzed using DAVID. P-values, FDRs and gene lists included in GO terms are summarized.
Please see also Supplementary figure 9.
Click here to Download Table S1 Click here to Download Table S2 Click here to Download    Enriched GO terms of mRNAs showing both upregulation and stabilization more than 1.5-fold (1), those showing both mRNA and pre-mRNA upregulation more than 1.5fold (2), and those showing both mRNA and pre-mRNA downregulation more than 1.5fold (3)