The CCR4-NOT complex suppresses untimely translational activation of maternal mRNAs.

Control of mRNA poly(A) tails is essential for regulation of mRNA metabolism, specifically translation efficiency and mRNA stability. Gene expression in maturing oocytes relies largely on post-transcriptional regulation, as genes are transcriptionally silent during oocyte maturation. The CCR4-NOT complex is a major mammalian deadenylase, which regulates poly(A) tails of maternal mRNAs; however, the function of the CCR4-NOT complex in translational regulation has not been well understood. Here, we show that this complex suppresses translational activity of maternal mRNAs during oocyte maturation. Oocytes lacking all CCR4-NOT deadenylase activity owing to genetic deletion of its catalytic subunits, Cnot7 and Cnot8, showed a large-scale gene expression change caused by increased translational activity during oocyte maturation. Developmental arrest during meiosis I in these oocytes resulted in sterility of oocyte-specific Cnot7 and Cnot8 knockout female mice. We further showed that recruitment of CCR4-NOT to maternal mRNAs is mediated by the 3'UTR element CPE, which suppresses translational activation of maternal mRNAs. We propose that suppression of untimely translational activation of maternal mRNAs via deadenylation by CCR4-NOT is essential for proper oocyte maturation.

The CCR4-NOT complex maintains liver homeostasis through mRNA deadenylation.

The biological significance of deadenylation in global gene expression is not fully understood. Here, we show that the CCR4-NOT deadenylase complex maintains expression of mRNAs, such as those encoding transcription factors, cell cycle regulators, DNA damage response-related proteins, and metabolic enzymes, at appropriate levels in the liver. Liver-specific disruption of Cnot1, encoding a scaffold subunit of the CCR4-NOT complex, leads to increased levels of mRNAs for transcription factors, cell cycle regulators, and DNA damage response-related proteins because of reduced deadenylation and stabilization of these mRNAs. CNOT1 suppression also results in an increase of immature, unspliced mRNAs (pre-mRNAs) for apoptosis-related and inflammation-related genes and promotes RNA polymerase II loading on their promoter regions. In contrast, mRNAs encoding metabolic enzymes become less abundant, concomitant with decreased levels of these pre-mRNAs. Lethal hepatitis develops concomitantly with abnormal mRNA expression. Mechanistically, the CCR4-NOT complex targets and destabilizes mRNAs mainly through its association with Argonaute 2 (AGO2) and butyrate response factor 1 (BRF1) in the liver. Therefore, the CCR4-NOT complex contributes to liver homeostasis by modulating the liver transcriptome through mRNA deadenylation.

RSK-MASTL Pathway Delays Meiotic Exit in Mouse Zygotes to Ensure Paternal Chromosome Stability.

During vertebrate fertilization, sperm chromatin remodeling occurs concomitantly with maternal chromosome segregation at anaphase II, leading to simultaneous formation of two pronuclei. In mammals, these processes take much longer than in other vertebrates. Here, we explore the molecular basis and physiological importance of this mammalian-specific temporal regulation using mouse oocytes. We demonstrate the involvement of protein phosphatase in temporal regulation. Early onset of pronuclear formation causes paternal-biased abnormalities in pronuclear morphology and chromosome segregation at the first mitosis. After oocyte activation, CDK1-MASTL-ENSA, a protein phosphatase 2A-suppressive pathway, remains active despite the absence of cyclin B and contributes to delayed pronuclear formation. Sustained activation of MASTL involves ribosomal S6 kinase (RSK)-mediated phosphorylation of Thr297, which is conserved only among mammalian MASTLs. Our findings reveal the role of RSK in mouse oocytes, showing that the RSK-MASTL pathway allows mammalian-specific prolonged meiotic exit and ensures the faithful conversion from sperm to paternal pronuclei.

The microtubule-binding and coiled-coil domains of Kid are required to turn off the polar ejection force at anaphase.

Mitotic chromosomes move dynamically along the spindle microtubules using the forces generated by motor proteins such as chromokinesin Kid (also known as KIF22). Kid generates a polar ejection force and contributes to alignment of the chromosome arms during prometaphase and metaphase, whereas during anaphase, Kid contributes to chromosome compaction. How Kid is regulated and how this regulation is important for chromosome dynamics remains unclear. Here, we address these questions by expressing mutant forms of Kid in Kid-deficient cells. We demonstrate that Cdk1-mediated phosphorylation of Thr463 is required to generate the polar ejection force on Kid-binding chromosomes, whereas dephosphorylation of Thr463 prevents generation of the ejection force on such chromosomes. In addition to activation of the second microtubule-binding domain through dephosphorylation of Thr463, the coiled-coil domain is essential in suspending generation of the polar ejection force, preventing separated chromosomes from becoming recongressed during anaphase. We propose that phosphorylation of Thr463 switches the mitotic chromosome movement from an anti-poleward direction to a poleward direction by converting the Kid functional mode from polar-ejection-force-ON to -OFF during the metaphase-anaphase transition, and that both the second microtubule-binding domain and the coiled-coil domain are involved in this switching process.

Inactivation of mitogen-activated protein kinase is neither necessary nor sufficient for the onset of pronuclear formation in mouse oocytes.

Mammalian oocytes are arrested at metaphase II due to high MAP kinase activity. After fertilization, oocytes resume meiosis, leading to female chromosome segregation, polar body emission and pronuclear (PN) formation. Previous biochemical studies showed that MAP kinase activity remained high for several hours after fertilization and began to decrease in parallel with PN formation. It has been thought that MAP kinase activity is incompatible with PN formation, and its inactivation is required for the initiation of PN formation in mammalian oocytes. In this study, we revisited this hypothesis by examining MAP kinase activity and PN formation in individual mouse oocytes using cytological analysis. We showed that MAP kinase activity in oocytes could be evaluated using phospho-ERK1/2 immunofluorescent staining. Co-immunofluorescent staining of phospho-ERK1/2 and nuclear pore components showed that PN formation preceded MAP kinase inactivation and could be initiated while MAP kinase activity was still high. Moreover, artificial inactivation of MAP kinase or its downstream target, ribosomal S6 kinase, accelerated but did not rapidly induce PN formation. Our results show that although the MAP kinase pathway negatively regulates PN formation, its inactivation is neither necessary nor sufficient for PN formation. These results suggest the involvement of other essential factor(s) in this process.