Essential functions of the CNOT7/8 catalytic subunits of the CCR4-NOT complex in mRNA regulation and cell viability

Shortening of mRNA poly(A) tails (deadenylation) to trigger their decay is mediated mainly by the CCR4-NOT deadenylase complex. While four catalytic subunits (CNOT6, 6L 7, and 8) have been identified in the mammalian CCR4-NOT complex, their individual biological roles are not fully understood. In this study, we addressed the contribution of CNOT7/8 to viability of primary mouse embryonic fibroblasts (MEFs). We found that MEFs lacking CNOT7/8 expression [Cnot7/8-double knockout (dKO) MEFs] undergo cell death, whereas MEFs lacking CNOT6/6L expression (Cnot6/6l-dKO MEFs) remain viable. Co-immunoprecipitation analyses showed that CNOT6/6L are also absent from the CCR4-NOT complex in Cnot7/8-dKO MEFs. In contrast, either CNOT7 or CNOT8 still interacts with other subunits in the CCR4-NOT complex in Cnot6/6l-dKO MEFs. Exogenous expression of a CNOT7 mutant lacking catalytic activity in Cnot7/8-dKO MEFs cannot recover cell viability, even though CNOT6/6L exists to some extent in the CCR4-NOT complex, confirming that CNOT7/8 is essential for viability. Bulk poly(A) tail analysis revealed that mRNAs with longer poly(A) tails are more numerous in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Consistent with elongated poly(A) tails, more mRNAs are upregulated and stabilized in Cnot7/8-dKO MEFs than in Cnot6/6l-dKO MEFs. Importantly, Cnot6/6l-dKO mice are viable and grow normally to adulthood. Taken together, the CNOT7/8 catalytic subunits are essential for deadenylation, which is necessary to maintain cell viability, whereas CNOT6/6L are not

Loss of β-cell identity and diabetic phenotype in mice caused by disruption of CNOT3-dependent mRNA deadenylation

Pancreatic β-cells are responsible for production and secretion of insulin in response to increasing blood glucose levels. Defects in β-cell function lead to hyperglycemia and diabetes mellitus. Here, we show that CNOT3, a CCR4-NOT deadenylase complex subunit, is dysregulated in islets in diabetic db/db mice, and that it is essential for murine β cell maturation and identity. Mice with β cell-specific Cnot3 deletion (Cnot3βKO) exhibit impaired glucose tolerance, decreased β cell mass, and they gradually develop diabetes. Cnot3βKO islets display decreased expression of key regulators of β cell maturation and function. Moreover, they show an increase of progenitor cell markers, β cell-disallowed genes, and genes relevant to altered β cell function. Cnot3βKO islets exhibit altered deadenylation and increased mRNA stability, partly accounting for the increased expression of those genes. Together, these data reveal that CNOT3-mediated mRNA deadenylation and decay constitute previously unsuspected post-transcriptional mechanisms essential for β cell identity

Neuronal XRN1 is required for maintenance of whole-body metabolic homeostasis

Control of mRNA stability and degradation is essential for appropriate gene expression, and its dysregulation causes various disorders, including cancer, neurodegenerative diseases, diabetes, and obesity. The 5′–3′ exoribonuclease XRN1 executes the last step of RNA decay, but its physiological impact is not well understood. To address this, forebrain-specific Xrn1 conditional knockout mice (Xrn1-cKO) were generated, as Xrn1 null mice were embryonic lethal. Xrn1-cKO mice exhibited obesity with leptin resistance, hyperglycemia, hyperphagia, and decreased energy expenditure. Obesity resulted from dysregulated communication between the central nervous system and peripheral tissues. Moreover, expression of mRNAs encoding proteins that regulate appetite and energy expenditure was dysregulated in the hypothalamus of Xrn1-cKO mice. Therefore, we propose that XRN1 function in the hypothalamus is critical for maintenance of metabolic homeostasis

Translational control of entrainment and synchrony of the suprachiasmatic circadian clock by mTOR/4E-BP1 signaling

SummaryProtein synthesis is critical for circadian clock function, but little is known of how translational regulation controls the master pacemaker in mammals, the suprachiasmatic nucleus (SCN). Here we demonstrate that the pivotal translational repressor, the eukaryotic translational initiation factor 4E binding protein 1 (4E-BP1), is rhythmically regulated via the mechanistic target of rapamycin (mTOR) signaling in the SCN and preferentially represses vasoactive intestinal peptide (Vip) mRNA translation. Knockout (KO) of Eif4ebp1 (gene encoding 4E-BP1) leads to upregulation of VIP and higher amplitude of molecular rhythms in the SCN. Consequently, the 4E-BP1 null mice exhibit accelerated re-entrainment to a shifted light/dark cycle and are more resistant to the rhythm-disruptive effects of constant light. Conversely, in Mtor+/− mice VIP expression is decreased and susceptibility to the effects of constant light is increased. These results reveal a key role for mTOR/4E-BP1-mediated translational control in regulating entrainment and synchrony of the master clock

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

Control of embryonic stem cells self-renewal and differentiation via coordinated splicing and translation of YY2

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Active-site mTOR inhibitors augment HSV1-dICP0 infection in cancer cells via dysregulated eIF4E/4E-BP axis

Herpes Simplex Virus 1 (HSV1) is amongst the most clinically advanced oncolytic virus platforms. However, efficient and sustained viral replication within tumours is limiting. Rapamycin can stimulate HSV1 replication in cancer cells, but active-site dual mTORC1 and mTORC2 (mammalian target of rapamycin complex 1 and 2) inhibitors (asTORi) were shown to suppress the virus in normal cells. Surprisingly, using the infected cell protein 0 (ICP0)-deleted HSV1 (HSV1-dICP0), we found that asTORi markedly augment infection in cancer cells and a mouse mammary cancer xenograft. Mechanistically, asTORi repressed mRNA translation in normal cells, resulting in defective antiviral response but also inhibition of HSV1-dICP0 replication. asTORi also reduced antiviral response in cancer cells, however in contrast to normal cells, transformed cells and cells transduced to elevate the expression of eukaryotic initiation factor 4E (eIF4E) or to silence the repressors eIF4E binding proteins (4E-BPs), selectively maintained HSV1-dICP0 protein synthesis during asTORi treatment, ultimately supporting increased viral replication. Our data show that altered eIF4E/4E-BPs expression can act to promote HSV1-dICP0 infection under prolonged mTOR inhibition. Thus, pharmacoviral combination of asTORi and HSV1 can target cancer cells displaying dysregulated eIF4E/4E-BPs axis.</div

Household income and the risk of incident hypertension in employees at multiple workplaces in Japan : J-HOPE

This cohort study aimed to investigate the association between household income and incident hypertension in a Japanese employed population. During 2012, a total of 4314 normotensive daytime employees (3153 men and 1161 women) were included in this study. Participants had a wide range of occupations and were employed at one of 12 workplaces from various economic sectors in Japan. After a 2-year follow-up, incident hypertension was compared among groups according to household income: = 10.0 million Japanese yen ( yen )/year. A Cox proportional hazard model was used to calculate the hazard ratio for incident hypertension in each household income group, compared with the group earning = 10.0 million yen /year after adjusting for age, baseline systolic blood pressure, worksite, type of occupation, number of family members, and smoking status. This positive relationship was attenuated but remained significant after further adjustment for alcohol consumption and body mass index, both of which were higher among men with higher household income. Conversely, there was no significant difference for women in the risk of incident hypertension among household income groups, although those with higher household income tended to have a lower risk of incident hypertension. Household income is positively associated with the onset of hypertension in Japanese employed men working daytime hours

CNOT7 Outcompetes Its Paralog CNOT8 for Integration into The CCR4-NOT Complex

The CCR4-NOT deadenylase complex is a major post-transcriptional regulator of eukaryotic gene expression. CNOT7 and CNOT8 are both vertebrate homologs of the yeast CCR4-NOT catalytic subunit Caf1. They are highly similar and are sometimes considered redundant, but Cnot7 and Cnot8 knockout mice exhibit different phenotypes, implying distinct physiological functions. In this study, we reveal a non-reciprocal effect of CNOT7 on CNOT8, in which CNOT8 protein is increased in the depletion of CNOT7 without corresponding changes in mRNA levels whereas CNOT7 is not affected by the loss of CNOT8. Cnot8 mRNA may be bound by the CCR4-NOT complex, suggesting that CCR4-NOT might directly regulate CNOT8 expression. Cnot8 mRNA is relatively unstable, but Cnot7 knockdown did not stabilize Cnot8 mRNA, nor did it increase translation. CNOT8 protein was also less stable than CNOT7. CNOT7 showed greater affinity than CNOT8 for the CCR4-NOT scaffold protein CNOT1 and was able to block CNOT8 from binding to CNOT1. Depletion of CNOT7 increased CNOT8 incorporation into the CCR4-NOT complex and stabilized CNOT8. These data suggest that CNOT7 is the dominant paralog in CCR4-NOT and that CNOT7 and CNOT8 protein stability is regulated in distinct ways