Akinori Takahashi

Multifunctional roles of the mammalian CCR4-NOT complex in physiological phenomena.

The carbon catabolite repression 4 (CCR4)–negative on TATA-less (NOT) complex serves as one of the major deadenylases of eukaryotes. Although it was originally identified and characterized in yeast, recent studies have revealed that the CCR4–NOT complex also exerts important functions in mammals, -including humans. However, there are some differences in the composition and functions of the CCR4–NOT complex between mammals and yeast. It is noteworthy that each subunit of the CCR4–NOT complex has unique, multifunctional roles and is responsible for various physiological phenomena. This heterogeneity and versatility of the CCR4–NOT complex makes an overall understanding of this complex difficult. Here, we describe the functions of each subunit of the mammalian CCR4–NOT complex and discuss the molecular mechanisms by which it regulates homeostasis in mammals. Furthermore, a possible link between the disruption of the CCR4–NOT complex and various diseases will be discussed. Finally, we propose that the analysis of mice with each CCR4–NOT subunit knocked out is an effective strategy for clarifying its complicated functions and networks in mammals.

The role of the CNOT1 subunit of the CCR4-NOT complex in mRNA deadenylation and cell viability

The human CCR4-NOT deadenylase complex consists of at least nine enzymatic and non-enzymatic subunits. Accumulating evidence suggests that the non-enzymatic subunits are involved in the regulation of mRNA deadenylation, although their precise roles remain to be established. In this study, we addressed the function of the CNOT1 subunit by depleting its expression in HeLa cells. Flow cytometric analysis revealed that the sub G1 fraction was increased in CNOT1-depleted cells. Virtually, the same level of the sub G1 fraction was seen when cells were treated with a mixture of siRNAs targeted against all enzymatic subunits, suggesting that CNOT1 depletion induces apoptosis by destroying the CCR4-NOT-associated deadenylase activity. Further analysis revealed that CNOT1 depletion leads to a reduction in the amount of other CCR4-NOT subunits. Importantly, the specific activity of the CNOT6L immunoprecipitates-associated deadenylase from CNOT1-depleted cells was less than that from control cells. The formation of P-bodies, where mRNA decay is reported to take place, was largely suppressed in CNOT1-depleted cells. Therefore, CNOT1 has an important role in exhibiting enzymatic activity of the CCR4-NOT complex, and thus is critical in control of mRNA deadenylation and mRNA decay. We further showed that CNOT1 depletion enhanced CHOP mRNA levels and activated caspase-4, which is associated with endoplasmic reticulum ER stress-induced apoptosis. Taken together, CNOT1 depletion structurally and functionally deteriorates the CCR4-NOTcomplex and induces stabilization of mRNAs, which results in the increment of translation causing ER stress-mediated apoptosis. We conclude that CNOT1 contributes to cell viability by securing the activity of the CCR4-NOT deadenylase.

Distinct expression patterns of the subunits of the CCR4-NOT deadenylase complex during neural development.

The stability of mRNA influences the dynamics of gene expression. The mammalian CCR4-NOT complex is associated with deadenylase activity, which shortens the mRNA poly(A) tail and thereby contributes to destabilization of mRNAs. The complex consists of at least nine subunits and predominantly forms a 2.0MDa protein complex in HeLa cells. Accumulating evidence suggests that the CCR4-NOT complex is involved in cell growth and survival; however, the regulatory mechanisms of its biological activity remain obscure. Here, we analyzed the expression levels of the subunits of the CCR4-NOT complex in various mouse tissues and found that they showed distinct expression patterns. CNOT6, 6L, 7, and 10 were expressed nearly ubiquitously, whereas others were expressed in tissue-specific manners, such as those displaying especially high expression in the brain. Furthermore, CNOT2, 3, 6, and 8 were rapidly downregulated during differentiation of neural stem cells. These findings suggest that subunit composition of the CCR4-NOT complex differs among tissues and is altered during neural development, thereby imparting an additional layer of specificity in the control of gene expression.

Tob2 inhibits peroxisome proliferator-activated receptor γ2 expression by sequestering Smads and C/EBPα during adipocyte differentiation.

ABSTRACT Adipogenesis is an important component of adipose tissue development and is critically related to obesity. A cascade of transcription factors is involved in adipogenesis, in which peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding proteins (C/EBPs) play pivotal roles. Bone morphogenetic proteins (BMPs) and Smad proteins are implicated in this cascade, although the precise regulatory mechanisms have yet to be elucidated. Here, we show that Tob2, a member of the Tob/BTG antiproliferative protein family, inhibits adipogenesis by interfering with Smad signaling. tob2 expression is downregulated in the white adipose tissue of high-fat diet-induced or genetically mutated obese mice. Consistent with this, tob2−/− mice exhibit increased adiposity with augmented expression of the genes encoding the type 1A BMP receptor (BMPR1A) and PPARγ2 as well as their target genes. We further show accelerated adipogenesis in primary tob2−/− preadipocytes. Furthermore, exogenously expressed Tob2 inhibits adipogenic differentiation of 3T3-L1 preadipocytes: the Tob2 protein suppresses PPARγ2 transcription by inhibiting BMP2-induced Smad1/5 phosphorylation through its interaction with Smad6 and by sequestering C/EBPα from the PPARγ2 promoter. Thus, Tob2 negatively regulates adipogenesis by inhibiting PPARγ2 expression.

Diversity changes in Cretaceous inoceramid bivalves of Japan

ABSTRACT Temporal species-diversity changes in Japanese Cretaceous inoceramid bivalves were analyzed from an extensive literature survey and statistical analysis, with the following results: (1) Species diversity increased gradually from the Upper Albian to Lower Campanian, and then dropped suddenly across the Lower/Upper Campanian (LCa/UCa) boundary; (2) There is no statistical correlation between ammonoid and inoceramid diversity changes in Japan, which must reflect the different ecologies of both groups; (3) Relatively high extinction ratios occurred at boundaries near Oceanic Anoxic Events (OAEs). The extinction events at the Albian/Cenomanian, Cenomanian/Turonian, and Turonian/Coniacian boundaries were potentially caused by OAE1d, 2 and the onset of OAE3, respectively. The drastic diversity decrease at the LCa/UCa boundary probably resulted from an abrupt and large-scale relative sea-level fall in the Yezo forearc basin; and (4) The pattern of diversity changes is similar to that of long-term (2nd-order) eustatic sea-level changes. The following hypotheses are presented as the cause of these phenomena: changes in shelf area, the primary inoceramid habitat, controlled their diversity, or changes in the Cretaceous outcrop area (rock volume) associated with sea-level changes controlled their diversity. It is possible that a combination of both factors controlled diversity patterns.

Involvement of CNOT3 in mitotic progression through inhibition of MAD1 expression

The stability of mRNA influences the dynamics of gene expression. The CCR4-NOT complex, the major deadenylase in mammalian cells, shortens the mRNA poly(A) tail and contributes to the destabilization of mRNAs. The CCR4-NOT complex plays pivotal roles in various physiological functions, including cell proliferation, apoptosis, and metabolism. Here, we show that CNOT3, a subunit of the CCR4-NOT complex, is involved in the regulation of the spindle assembly checkpoint, suggesting that the CCR4-NOT complex also plays a part in the regulation of mitosis. CNOT3 depletion increases the population of mitotic-arrested cells and specifically increases the expression of MAD1 mRNA and its protein product that plays a part in the spindle assembly checkpoint. We showed that CNOT3 depletion stabilizes the MAD1 mRNA, and that MAD1 knockdown attenuates the CNOT3 depletion-induced increase of the mitotic index. Basing on these observations, we propose that CNOT3 is involved in the regulation of the spindle assembly checkpoint through its ability to regulate the stability of MAD1 mRNA.

The CCR4-NOT deadenylase complex controls Atg7-dependent cell death and heart function

Destabilization of Atg7 mRNA by the CCR4-NOT complex prevents p53-dependent cell death in the heart. Protecting the heart by destabilizing mRNA The removal of polyadenylate tails from mRNAs by the CCR4-NOT complex marks these mRNAs for degradation. Yamaguchi et al. (see also the Focus by Das) found that this activity of this complex was required to prevent cell death in the heart. Mice deficient in a component of this complex suffered from cardiac dysfunction and died of heart failure due to cardiomyocyte death. The CCR4-NOT complex deadenylated Atg7 mRNA, which encodes a protein required for autophagy, a process by which cellular constituents and organelles are digested. The increase in Atg7 in the mutant mice resulted in activation of cell death–associated genes by the transcription factor p53. Drugs that increase autophagy have been explored for the treatment of various diseases, but the authors note that their results raise the possibility of cardiovascular side effects for such drugs. Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4–negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death–promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis.

The CCR4-NOT deadenylase activity contributes to generation of induced pluripotent stem cells

Somatic cells can be reprogrammed as induced pluripotent stem cells (iPSCs) by introduction of the transcription factors, OCT3/4, KLF4, SOX2, and c-MYC. The CCR4-NOT complex is the major deadenylase in eukaryotes. Its subunits Cnot1, Cnot2, and Cnot3 maintain pluripotency and self-renewal of mouse and human embryonic stem (ES) cells and contribute to the transition from partial to full iPSCs. However, little is known about how the CCR4-NOT complex post-transcriptionally regulates the reprogramming process. Here, we show that the CCR4-NOT deadenylase subunits Cnot6, Cnot6l, Cnot7, and Cnot8, participate in regulating iPSC generation. Cnot1 knockdown suppresses expression levels of Cnot6, Cnot6l, Cnot7, and Cnot8 in mouse embryonic fibroblasts (MEFs) and decreases the number of alkaline phosphatase (ALP)-positive colonies after iPSC induction. Intriguingly, Cnot1 depletion allows Eomes and p21 mRNAs to persist, increasing their expression levels. Both mRNAs have longer poly(A) tails in Cnot1-depleted cells. Conversely, forced expression of a combination of Cnot6, Cnot6l, Cnot7, and Cnot8 increases the number of ALP-positive colonies after iPSC induction and decreases expression levels of Eomes and p21 mRNAs. Based on these observations, we propose that the CCR4-NOT deadenylase activity contributes to iPSC induction.

Adipocyte‐specific disruption of mouse Cnot3 causes lipodystrophy

Lipodystrophy involves a loss of adipose tissue. In mice, disruption of adipose tissue Cnot3, a subunit of the CCR4‐NOT deadenylase complex, causes adipose tissue anomalies. In Cnot3ad−/− mice, white adipose tissue (WAT) decreases concomitantly with enhanced inflammation, whereas brown adipose tissue increases and contains larger lipid droplets. Cnot3ad−/− mice show hyperinsulinemia, hyperglycemia, insulin resistance, and glucose intolerance, and cannot maintain body temperature during cold exposure. Increased expression of inflammatory genes and decreased leptin expression also occur in Cnot3ad−/− WAT, achieving levels similar to those in lipodystrophic aP2‐nSrebp1c and Ppargldi/+ mice; thus, Cnot3ad−/− mice exhibit lipodystrophy.