Insufficient liver maturation affects murine early postnatal hair cycle

Abnormal hair loss results from a variety of factors, such as metabolic dysfunctions, immunodeficiency, and environmental stressors. Here, we report that mutant mice having defects in liver function, develop alopecia. We have shown previously that in mice lacking a Cnot3 gene, which encodes an essential component of the CCR4-NOT deadenylase complex in liver (Cnot3-LKO mice), the liver does not mature properly, resulting in various pathologies such as hepatitis, hepatic necrosis, and anemia. Unexpectedly, Cnot3-LKO mice start to lose hair around postnatal day 17 (P17). The region of hair loss expands all across their backs and symptoms persist until around P28-30. Afterward, hair re-grows, and Cnot3-LKO mice show complete hair recovery by P40. The phenotype is dependent on mouse genotype, indicating that hair follicle morphogenesis and cycling are influenced by abnormal liver development. By performing histological, quantitative PCR, and immunoblot analyses, we detected sebaceous gland (SG) hypertrophy accompanied by an increase of peroxisome proliferator-activated receptor gamma (PPARgamma). Collectively, these findings suggest that paracrine signaling related to liver function influences hair growth, at least in part, by altering lipid metabolism

CNOT1 regulates circadian behaviour through Per2 mRNA decay in a deadenylation-dependent manner

Circadian clocks are an endogenous internal timekeeping mechanism that drives the rhythmic expression of genes, controlling the 24 h oscillatory pattern in behaviour and physiology. It has been recently shown that post-transcriptional mechanisms are essential for controlling rhythmic gene expression. Controlling the stability of mRNA through poly(A) tail length modulation is one such mechanism. In this study, we show that Cnot1, encoding the scaffold protein of the CCR4-NOT deadenylase complex, is highly expressed in the suprachiasmatic nucleus, the master timekeeper. CNOT1 deficiency in mice results in circadian period lengthening and alterations in the mRNA and protein expression patterns of various clock genes, mainly Per2. Per2 mRNA exhibited a longer poly(A) tail and increased mRNA stability in Cnot1+/− mice. CNOT1 is recruited to Per2 mRNA through BRF1 (ZFP36L1), which itself oscillates in antiphase with Per2 mRNA. Upon Brf1 knockdown, Per2 mRNA is stabilized leading to increased PER2 expression levels. This suggests that CNOT1 plays a role in tuning and regulating the mammalian circadian clock.journal articl

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

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

CNOT3 targets negative cell cycle regulators in non-small cell lung cancer development

Lung cancer is one of the major causes of cancer death and clarification of its molecular pathology is highly prioritized. The physiological importance of mRNA degradation through the CCR4-NOT deadenylase has recently been highlighted. For example, mutation in CNOT3, a gene coding for CNOT3 subunit of the CCR4-NOT complex, is found to be associated with T-cell acute lymphoblastic leukemia, T-ALL, though its contribution to other cancers has not been reported. Here, we provide evidence suggesting that CNOT3 is required for the growth of non-small cell lung cancer. Depletion of CNOT3 suppresses proliferation of A549 human non-small cell lung cancer cells with enhanced mRNA stability and subsequent elevated expression of p21. In addition, we identified the mRNA for Kruppel-like factor 2 transcription factor, an inducer of p21, as a novel mRNA degradation target of CNOT3 in non-small cell lung cancer cells. Aberrant up-regulation of Kruppel-like factor 2 by CNOT3 depletion leads to impairment in the proliferation of A549 cells. Consistent with these findings, elevated mRNA expression of CNOT3 in non-small cell lung cancer in comparison with the paired normal lung epithelium was confirmed through scrutinization of the RNA-sequencing datasets from The Cancer Genome Atlas. Moreover, we found an inverse correlation between CNOT3 and CDKN1A (encoding p21) mRNA expression using the combined datasets of normal lung epithelium and non-small cell lung cancer. Thus, we propose that the up-regulation of CNOT3 facilitates the development of non-small cell lung cancer through down-regulation of Kruppel-like factor 2 and p21, contrary to tumor suppressive functions of CNOT3 in T-ALL

Regulation of CCR4-NOT complex deadenylase activity and cellular responses by MK2-dependent phosphorylation of CNOT2

CCR4-NOT complex-mediated mRNA deadenylation serves critical functions in multiple biological processes, yet how this activity is regulated is not fully understood. Here, we show that osmotic stress induces MAPKAPK-2 (MK2)-mediated phosphorylation of CNOT2. Programmed cell death is greatly enhanced by osmotic stress in CNOT2-depleted cells, indicating that CNOT2 is responsible for stress resistance of cells. Although wild-type (WT) and non-phosphorylatable CNOT2 mutants reverse this sensitivity, a phosphomimetic form of CNOT2, in which serine at the phosphorylation site is replaced with glutamate, does not have this function. We also show that mRNAs have elongated poly(A) tails in CNOT2-depleted cells and that introduction of CNOT2 WT or a non-phosphorylatable mutant, but not phosphomimetic CNOT2, renders their poly(A) tail lengths comparable to those in control HeLa cells. Consistent with this, the CCR4-NOT complex containing phosphomimetic CNOT2 exhibits less deadenylase activity than that containing CNOT2 WT. These data suggest that CCR4-NOT complex deadenylase activity is regulated by post-translational modification, yielding dynamic control of mRNA deadenylation

The CCR4–NOT deadenylase complex safeguards thymic positive selection by down-regulating aberrant pro-apoptotic gene expression

A repertoire of T cells with diverse antigen receptors is selected in the thymus. However, detailed mechanisms underlying this thymic positive selection are not clear. Here we show that the CCR4-NOT complex limits expression of specific genes through deadenylation of mRNA poly(A) tails, enabling positive selection. Specifically, the CCR4-NOT complex is up-regulated in thymocytes before initiation of positive selection, where in turn, it inhibits up-regulation of pro-apoptotic Bbc3 and Dab2ip. Elimination of the CCR4-NOT complex permits up-regulation of Bbc3 during a later stage of positive selection, inducing thymocyte apoptosis. In addition, CCR4-NOT elimination up-regulates Dab2ip at an early stage of positive selection. Thus, CCR4-NOT might control thymocyte survival during two-distinct stages of positive selection by suppressing expression levels of pro-apoptotic molecules. Taken together, we propose a link between CCR4-NOT-mediated mRNA decay and T cell selection in the thymus

Appetitive Olfactory Learning and Long-Term Associative Memory in Caenorhabditis elegans

Because of the relative simplicity of its nervous system, Caenorhabditis elegans is a useful model organism to study learning and memory at cellular and molecular levels. For appetitive conditioning in C. elegans, food has exclusively been used as an unconditioned stimulus (US). It may be difficult to analyze neuronal circuits for associative memory since food is a multimodal combination of olfactory, gustatory, and mechanical stimuli. Here, we report classical appetitive conditioning and associative memory in C. elegans, using 1-nonanol as a conditioned stimulus (CS), and potassium chloride (KCl) as a US. Before conditioning, C. elegans innately avoided 1-nonanol, an aversive olfactory stimulus, and was attracted by KCl, an appetitive gustatory stimulus, on assay agar plates. Both massed training without an intertrial interval (ITI) and spaced training with a 10-min ITI induced significant levels of memory of association regarding the two chemicals. Memory induced by massed training decayed within 6 h, while that induced by spaced training was retained for more than 6 h. Animals treated with inhibitors of transcription or translation formed the memory induced by spaced training less efficiently than untreated animals, whereas the memory induced by massed training was not significantly affected by such treatments. By definition, therefore, memories induced by massed training and spaced training are classified as short-term memory (STM) and long-term memory (LTM), respectively. When animals conditioned by spaced training were exposed to 1-nonanol alone, their learning index was lower than that of untreated animals, suggesting that extinction learning occurs in C. elegans. In support of these results, C. elegans mutants defective in nmr-1, encoding an NMDA receptor subunit, formed both STM and LTM less efficiently than wild-type animals, while mutations in crh-1, encoding a ubiquitous transcription factor CREB required for memory consolidation, affected LTM, but not STM. The paradigm established in the present study should allow us to elucidate neuronal circuit plasticity for appetitive learning and memory in C. elegans

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