CCR4-NOT Complex Research Articles

CNOT1 Microrna Single Nucleotide Polymorphism (SNP) Determines the Occurrence of Methotrexate Related Mucositis in Pediatric Acute Lymphoblastic Leukemia

BACKGROUNDCure rates of pediatric acute lymphoblastic leukemia (ALL) have reached 90% in the developed countries. However, toxicity due to chemotherapeutic regimens occurs frequently but with great heterogeneity. This suggests that genetic variation is involved. In order to identify determinants of adverse effects, recent studies have investigated pharmacogenetic features in relation to toxicity. Most of these studies examined coding regions of the genome. Recently, it has been described that epi genetic regulators, such as micro-RNA's (miRNA), might also have an important regulatory function in genes involved in drug related toxicity. In a recent study 25 miRNA SNPs were found to be related to toxicity in pediatric ALL treatment (Lopez-Lopez, PLoS ONE, 2014). In pediatric ALL mucositis is one of the most frequent side effects during high dose methotrexate (MTX) treatment.AIMThe aim of this study was to detect novel, epigenetic biomarkers that predict MTX related oral mucositis in pediatric acute lymphoblastic leukemia (B- and T cell) by studying single nucleotide polymorphisms (SNP) involved in miRNA levels and function.METHODSDNA was isolated from whole blood of 118 pediatric ALL patients that were treated with high dose MTX (5 gr/m2) according to the Dutch Childhood Oncology Group ALL-10 protocol. The recently published 25 SNPs, involved in miRNA function and located in the DROSHA, CNOT1, CNOT4, EIF2C1, GEMIN3, GEMIN4, MIR604, MIR453, MIR2110, MIR2053, MIR1294, MIR1206, DICER, XPO5 and TNRC6B genes, were selected for genotyping. Toxicity data during the consolidation phase were prospectively collected and documented according to the National Cancer Institute (NCI) v.3.0 score system. Mucositis NCI grade ≥ 3 (grade 3: confluent ulcerations, bleeding with minor trauma), was considered as clinical significant toxicity and was used as endpoint.RESULTSMucositis was the only recurring toxicity in this prospectively well-documented cohort and therefore used as endpoint of this study. A selection of 20 of the previously identified 25 candidate SNPs was studied based on technical feasibility. In addition, 1 SNP in the XPO 5 gene was not considered for analysis because it was not in Hardy Weinberg equilibrium. Mucositis occurred in 19% of the patients in at least one of the MTX courses. Only the TT genotype of rs11866002 in the CNOT1 (CCR4-NOT complex, subunit 1) gene was associated with a higher risk of developing mucositis (NCI ≥ 3) compared to carries of CC/CT. The other 18 candidate SNPs analyzed did not show statistically significant associations.CONCLUSIONThe inter-patient variability of mucosal toxicity was not associated with most of our investigated SNPs which are involved in miRNA transcription and function. CNOT1 rs11866002 C>T was the only single nucleotide polymorphism associated with the occurrence of oral mucositis during pediatric acute lymphoblastic leukemia treatment.We acknowledge the Foundation Children Cancerfree (KiKa), Amstelveen, The Netherlands, for funding this research. DisclosuresNo relevant conflicts of interest to declare.

Post-transcriptional modulation of interleukin 8 by CNOT6L regulates skeletal muscle differentiation

CNOT6L is a deadenylase subunit belonging to the CCR4-NOT complex, a major deadenylase complex in eukaryotes involved at multiple levels in regulation of gene expression. While CNOT6L is expressed in skeletal muscle cells, its specific functions in this tissue are still largely unknown. Our previous work highlighted the functional of CNOT6L in skeletal muscle cell differentiation. To further explore how CNOT6L regulates myogenesis, we used here gene expression analysis to identify CNOT6L mRNA targets in human myoblasts. Among these novel targets, IL-8 (interleukin 8) mRNA was the most upregulated in CNOT6L knock-down (KD) cells. Biochemical approaches and poly (A) tail length assays showed that IL-8 mRNA is a direct target of CNOT6L, and further investigations by loss- and gain-of-function assays pointed out that IL-8 is an important effector of myogenesis. Therefore, we have characterized CNOT6L-IL-8 as a new signaling axis that regulates myogenesis.

Dead end1 is an essential partner of NANOS2 for selective binding of target RNAs in male germ cell development.

RNA-binding proteins (RBPs) play important roles for generating various cell types in many developmental processes, including eggs and sperms. Nanos is widely known as an evolutionarily conserved RNA-binding protein implicated in germ cell development. Mouse NANOS2 interacts directly with the CCR4-NOT (CNOT) deadenylase complex, resulting in the suppression of specific RNAs. However, the mechanisms involved in target specificity remain elusive. We show that another RBP, Dead end1 (DND1), directly interacts with NANOS2 to load unique RNAs into the CNOT complex. This interaction is mediated by the zinc finger domain of NANOS2, which is essential for its association with target RNAs. In addition, the conditional deletion of DND1 causes the disruption of male germ cell differentiation similar to that observed in Nanos2-KO mice. Thus, DND1 is an essential partner for NANOS2 that leads to the degradation of specific RNAs. We also present the first evidence that the zinc finger domain of Nanos acts as a protein-interacting domain for another RBP, providing a novel insight into Nanos-mediated germ cell development.

Twin Promotes the Maintenance and Differentiation of Germline Stem Cell Lineage through Modulation of Multiple Pathways.

The central question in stem cell regulation is how the balance between self-renewal and differentiation is controlled at the molecular level. This study uses germline stem cells (GSCs) in the Drosophila ovary to demonstrate that the Drosophila CCR4 homologTwin is required intrinsically to promote both GSCself-renewal and progeny differentiation. Twin/CCR4 is one of the two catalytic subunits in the highly conserved CCR4-NOT mRNA deadenylase complex. Twin works within the CCR4-NOT complex to intrinsically maintain GSC self-renewal, at least partly by sustaining E-cadherin-mediated GSC-niche interaction and preventing transposable element-induced DNA damage. It promotes GSC progeny differentiation by forming protein complexes with differentiation factors Bam and Bgcn independently of other CCR4-NOT components. Interestingly, Bam can competitively inhibit the association of Twin with Pop2 in the CCR4-NOT complex. Therefore, this study demonstrates that Twin has important intrinsic roles in promoting GSC self-renewal and progeny differentiation by functioning in different protein complexes.

CNOT3 suppression promotes necroptosis by stabilizing mRNAs for cell death-inducing proteins.

The CCR4-NOT complex is conserved in eukaryotes and is involved in mRNA metabolism, though its molecular physiological roles remain to be established. We show here that CNOT3-depleted mouse embryonic fibroblasts (MEFs) undergo cell death. Levels of other complex subunits are decreased in CNOT3-depleted MEFs. The death phenotype is rescued by introduction of wild-type (WT), but not mutated CNOT3, and is not suppressed by the pan-caspase inhibitor, zVAD-fluoromethylketone. Gene expression profiling reveals that mRNAs encoding cell death-related proteins, including receptor-interacting protein kinase 1 (RIPK1) and RIPK3, are stabilized in CNOT3-depleted MEFs. Some of these mRNAs bind to CNOT3, and in the absence of CNOT3 their poly(A) tails are elongated. Inhibition of RIPK1-RIPK3 signaling by a short-hairpin RNA or a necroptosis inhibitor, necrostatin-1, confers viability upon CNOT3-depleted MEFs. Therefore, we conclude that CNOT3 targets specific mRNAs to prevent cells from being disposed to necroptotic death.

Structure of a Human 4E-T/DDX6/CNOT1 Complex Reveals the Different Interplay of DDX6-Binding Proteins with the CCR4-NOT Complex.

The DEAD-box protein DDX6 is a central component of translational repression mechanisms in maternal mRNA storage in oocytes and microRNA-mediated silencing in somatic cells. DDX6 interacts with the CCR4-NOT complex and functions in concert with several post-transcriptional regulators, including Edc3, Pat1, and 4E-T. We show that the conserved CUP-homology domain (CHD) of human 4E-T interacts directly with DDX6 in both the presence and absence of the central MIF4G domain of CNOT1. The 2.1-Å resolution structure of the corresponding ternary complex reveals how 4E-T CHD wraps around the RecA2 domain of DDX6 and contacts CNOT1. Although 4E-T CHD lacks recognizable sequence similarity with Edc3 or Pat1, it shares the same DDX6-binding surface. In contrast to 4E-T, however, the Edc3 and Pat1 FDF motifs dissociate from DDX6 upon CNOT1 MIF4G binding in vitro. The results underscore the presence of a complex network of simultaneous and/or mutually exclusive interactions in DDX6-mediated repression.

Role of Ccr4-Not complex in heterochromatin formation at meiotic genes and subtelomeres in fission yeast.

BackgroundHeterochromatin is essential for chromosome segregation, gene silencing and genome integrity. The fission yeast Schizosaccharomyces pombe contains heterochromatin at centromeres, subtelomeres, and mating type genes, as well as at small islands of meiotic genes dispersed across the genome. This heterochromatin is generated by partially redundant mechanisms, including the production of small interfering RNAs (siRNAs) that are incorporated into the RITS protein complex (RNAi-Induced Transcriptional Silencing). The assembly of heterochromatin islands requires the function of the RNA-binding protein Mmi1, which recruits RITS to its mRNA targets and to heterochromatin islands. In addition, Mmi1 directs its targets to an exosome-dependent RNA elimination pathway.ResultsCcr4-Not is a conserved multiprotein complex that regulates gene expression at multiple levels, including RNA degradation and translation. We show here that Ccr4-Not is recruited by Mmi1 to its RNA targets. Surprisingly, Ccr4 and Caf1 (the mRNA deadenylase catalytic subunits of the Ccr4-Not complex) are not necessary for the degradation or translation of Mmi1 RNA targets, but are essential for heterochromatin integrity at Mmi1-dependent islands and, independently of Mmi1, at subtelomeric regions. Both roles require the deadenylase activity of Ccr4 and the Mot2/Not4 protein, a ubiquitin ligase that is also part of the complex. Genetic evidence shows that Ccr4-mediated silencing is essential for normal cell growth, indicating that this novel regulation is physiologically relevant. Moreover, Ccr4 interacts with components of the RITS complex in a Mmi1-independent manner.ConclusionsTaken together, our results demonstrate that the Ccr4-Not complex is required for heterochromatin integrity in both Mmi1-dependent and Mmi1-independent pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0018-4) contains supplementary material, which is available to authorized users.

CNOT3 contributes to early B cell development by controlling Igh rearrangement and p53 mRNA stability

The CCR4-NOT deadenylase complex plays crucial roles in mRNA decay and translational repression induced by poly(A) tail shortening. Although the in vitro activities of each component of this complex have been well characterized, its in vivo role in immune cells remains unclear. Here we show that mice lacking the CNOT3 subunit of this complex, specifically in B cells, have a developmental block at the pro- to pre-B cell transition. CNOT3 regulated generation of germline transcripts in the VH region of the immunoglobulin heavy chain (Igh) locus, compaction of the locus, and subsequent Igh gene rearrangement and destabilized tumor suppressor p53 mRNA. The developmental defect in the absence of CNOT3 could be partially rescued by ablation of p53 or introduction of a pre-rearranged Igh transgene. Thus, our data suggest that the CCR4-NOT complex regulates B cell differentiation by controlling Igh rearrangement and destabilizing p53 mRNA.

Telomeric repeat silencing in germ cells is essential for early development in Drosophila.

The germline-specific role of telomeres consists of chromosome end elongation and proper chromosome segregation during early developmental stages. Despite the crucial role of telomeres in germ cells, little is known about telomere biology in the germline. We analyzed telomere homeostasis in the Drosophila female germline and early embryos. A novel germline-specific function of deadenylase complex Ccr4-Not in the telomeric transcript surveillance mechanism is reported. Depletion of Ccr4-Not complex components causes strong derepression of the telomeric retroelement HeT-A in the germ cells, accompanied by elongation of the HeT-A poly(A) tail. Dysfunction of transcription factors Woc and Trf2, as well as RNA-binding protein Ars2, also results in the accumulation of excessively polyadenylated HeT-A transcripts in ovaries. Germline knockdowns of Ccr4-Not components, Woc, Trf2 and Ars2, lead to abnormal mitosis in early embryos, characterized by chromosome missegregation, centrosome dysfunction and spindle multipolarity. Moreover, the observed phenotype is accompanied by the accumulation of HeT-A transcripts around the centrosomes in early embryos, suggesting the putative relationship between overexpression of telomeric transcripts and mitotic defects. Our data demonstrate that Ccr4-Not, Woc, Trf2 and Ars2, components of different regulatory pathways, are required for telomere protection in the germline in order to guarantee normal development.

Ccr4-Not and TFIIS Function Cooperatively To Rescue Arrested RNA Polymerase II

Expression of the genome requires RNA polymerase II (RNAPII) to transcribe across many natural and unnatural barriers, and this transcription across barriers is facilitated by protein complexes called elongation factors (EFs). Genetic studies in Saccharomyces cerevisiae yeast suggest that multiple EFs collaborate to assist RNAPII in completing the transcription of genes, but the molecular mechanisms of how they cooperate to promote elongation are not well understood. The Ccr4-Not complex participates in multiple steps of mRNA metabolism and has recently been shown to be an EF. Here we describe how Ccr4-Not and TFIIS cooperate to stimulate elongation. We find that Ccr4-Not and TFIIS mutations show synthetically enhanced phenotypes, and biochemical analyses indicate that Ccr4-Not and TFIIS work synergistically to reactivate arrested RNAPII. Ccr4-Not increases the recruitment of TFIIS into elongation complexes and enhances the cleavage of the displaced transcript in backtracked RNAPII. This is mediated by an interaction between Ccr4-Not and the N terminus of TFIIS. In addition to revealing insights into how these two elongation factors cooperate to promote RNAPII elongation, our study extends the growing body of evidence suggesting that the N terminus of TFIIS acts as a docking/interacting site that allows it to synergize with other EFs to promote RNAPII transcription.

Xenopus CAF1 requires NOT1-mediated interaction with 4E-T to repress translation in vivo.

RNA-regulatory factors bound to 3′ UTRs control translation and stability. Repression often is associated with poly(A) removal. The deadenylase CAF1 is a core component of the CCR4–NOT complex. Our prior studies established that CAF1 represses translation independent of deadenylation. We sought the mechanism of its deadenylation-independent repression in Xenopus oocytes. Our data reveal a chain of interacting proteins that links CAF1 to CCR4–NOT and to Xp54 and 4E-T. Association of CAF1 with NOT1, the major subunit of CCR4–NOT, is required for repression by CAF1 tethered to a reporter mRNA. Affinity purification-mass spectrometry and coimmunoprecipitation revealed that at least five members of the CCR4–NOT complex were recruited by CAF1. The recruitment of these proteins required NOT1, as did the ability of tethered CAF1 to repress translation. In turn, NOT1 was needed to recruit Xp54 and 4E-T. We examined the role of 4E-T in repression using mutations that disrupted either eIF4E-dependent or -independent mechanisms. Expression of a 4E-T truncation that still bound eIF4E alleviated repression by tethered CAF1, NOT1, and Xp54. In contrast, a mutant 4E-T that failed to bind eIF4E did not. Repression of global translation was affected only by the eIF4E-dependent mechanism. Reporters bearing IRES elements revealed that repression via tethered CAF1 and Xp54 is cap- and eIF4E-independent, but requires one or more of eIF4A, eIF4B, and eIF4G. We propose that RNA-binding proteins, and perhaps miRNAs, repress translation through an analogous chain of interactions that begin with the 3′ UTR-bound repressor and end with the noncanonical activity of 4E-T.

Not4-dependent translational repression is important for cellular protein homeostasis in yeast.

Translation of aberrant or problematic mRNAs can cause ribosome stalling which leads to the production of truncated or defective proteins. Therefore, cells evolved cotranslational quality control mechanisms that eliminate these transcripts and target arrested nascent polypeptides for proteasomal degradation. Here we show that Not4, which is part of the multifunctional Ccr4-Not complex in yeast, associates with polysomes and contributes to the negative regulation of protein synthesis. Not4 is involved in translational repression of transcripts that cause transient ribosome stalling. The absence of Not4 affected global translational repression upon nutrient withdrawal, enhanced the expression of arrested nascent polypeptides and caused constitutive protein folding stress and aggregation. Similar defects were observed in cells with impaired mRNA decapping protein function and in cells lacking the mRNA decapping activator and translational repressor Dhh1. The results suggest a role for Not4 together with components of the decapping machinery in the regulation of protein expression on the mRNA level and emphasize the importance of translational repression for the maintenance of proteome integrity.

Protein Degradation of RNA Polymerase II-Association Factor 1(PAF1) Is Controlled by CNOT4 and 26S Proteasome.

The PAF complex (PAFc) participates in various steps of the transcriptional process, from initiation to termination, by interacting with and recruiting various proteins to the proper locus for each step. PAFc is an evolutionarily conserved, multi-protein complex comprising PAF1, CDC73, CTR9, LEO1, yRTF1 and, in humans, hSKI8. These components of PAFc work together, and their protein levels are closely interrelated. In the present study, we investigated the mechanism of PAF1 protein degradation. We found that PAF1 protein levels are negatively regulated by the expression of CNOT4, an ortholog of yNOT4 and a member of the CCR4-NOT complex. CNOT4 specifically controls PAF1 but not other components of PAFc at the protein level by regulating the polyubiquitination of PAF1 and its subsequent degradation by the 26S proteasome. The degradation of PAF1 was found to require nuclear localization, as no PAF1 degradation by CNOT4 and the 26S proteasome was observed with NLS (nucleus localization signal)-deficient PAF1 mutants. However, chromatin binding by PAF1 was not necessary for 26S proteasome- or CNOT4-mediated degradation. Our results suggest that CNOT4 controls the degradation of chromatin-unbound PAF1 via the 26S proteasome.

Architecture of the Ubiquitylation Module of the Yeast Ccr4-Not Complex

SummaryThe Ccr4-Not complex regulates eukaryotic gene expression at multiple levels, including mRNA turnover, translational repression, and transcription. We have studied the ubiquitylation module of the yeast Ccr4-Not complex and addressed how E3 ligase binds cognate E2 and how it is tethered to the complex. The 2.8-Å resolution crystal structure of the N-terminal RING domain of Not4 in complex with Ubc4 shows the detailed interactions of this E3-E2 complex. The 3.6-Å resolution crystal structure of the C-terminal domain of the yeast Not4 in complex with the C-terminal domain of Not1 reveals how a largely extended region at the C-terminus of Not4 wraps around a HEAT-repeat region of Not1. This C-terminal region of Not4 is only partly conserved in metazoans, rationalizing its weaker Not1-binding properties. The structural and biochemical data show how Not1 can incorporate both the ubiquitylation module and the Not2-Not3/5 module concomitantly in the Ccr4-Not complex.

Exploring the Requirement for the CCR4‐NOT Deadenylase Complex Subunit Regena/NOT2 in MicroRNA‐mediated Gene Silencing

A forward genetic screen was carried out using Drosophila melanogaster to determine the cellular components and molecular mechanisms that regulate microRNA (miRNA)‐mediated gene silencing. MiRNAs are non‐coding RNA molecules that interact with target‐gene messenger RNA transcripts via complementary base pairing, and silence target gene expression via interaction with the miRNA‐induced silencing complex (miRISC). Our screen utilized a Green Fluorescent Protein (GFP)‐based reporter to visualize miRNA‐mediated gene silencing. The GFP transgene contains miRNA binding sites, and is regulated by endogenous miRNAs. MiRNA‐mediated silencing is accomplished through translation block, mRNA degradation, or a combination of these mechanisms by miRISC. A requirement for the CCR4‐NOT complex subunit Regena/NOT2 in miRNA‐mediated gene silencing was discovered through genetic analysis of an isolated mutant. We are exploring the specific role of Regena/NOT2 in silencing by determining whether this CCR4‐NOT complex component is required for translational block, transcript degradation, or both. We are further characterizing a putative role for Regena/NOT2 in miRNA biogenesis and maturation, as has been demonstrated in Arabidopsis. Ongoing studies of additional mutants generated in the screen aim to identify the components of miRISC in different cellular contexts and with respect to different miRNA targets, to elucidate the complex molecular mechanisms of miRNA‐mediated silencing.

Ccr4-not regulates RNA polymerase I transcription and couples nutrient signaling to the control of ribosomal RNA biogenesis.

Ribosomal RNA synthesis is controlled by nutrient signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. mTORC1 regulates ribosomal RNA expression by affecting RNA Polymerase I (Pol I)-dependent transcription of the ribosomal DNA (rDNA) but the mechanisms involved remain obscure. This study provides evidence that the Ccr4-Not complex, which regulates RNA Polymerase II (Pol II) transcription, also functions downstream of mTORC1 to control Pol I activity. Ccr4-Not localizes to the rDNA and physically associates with the Pol I holoenzyme while Ccr4-Not disruption perturbs rDNA binding of multiple Pol I transcriptional regulators including core factor, the high mobility group protein Hmo1, and the SSU processome. Under nutrient rich conditions, Ccr4-Not suppresses Pol I initiation by regulating interactions with the essential transcription factor Rrn3. Additionally, Ccr4-Not disruption prevents reduced Pol I transcription when mTORC1 is inhibited suggesting Ccr4-Not bridges mTORC1 signaling with Pol I regulation. Analysis of the non-essential Pol I subunits demonstrated that the A34.5 subunit promotes, while the A12.2 and A14 subunits repress, Ccr4-Not interactions with Pol I. Furthermore, ccr4Δ is synthetically sick when paired with rpa12Δ and the double mutant has enhanced sensitivity to transcription elongation inhibition suggesting that Ccr4-Not functions to promote Pol I elongation. Intriguingly, while low concentrations of mTORC1 inhibitors completely inhibit growth of ccr4Δ, a ccr4Δ rpa12Δ rescues this growth defect suggesting that the sensitivity of Ccr4-Not mutants to mTORC1 inhibition is at least partially due to Pol I deregulation. Collectively, these data demonstrate a novel role for Ccr4-Not in Pol I transcriptional regulation that is required for bridging mTORC1 signaling to ribosomal RNA synthesis.

Multi-functional regulation of 4E-BP gene expression by the Ccr4-Not complex.

The mechanistic target of rapamycin (mTOR) signaling pathway is highly conserved from yeast to humans. It senses various environmental cues to regulate cellular growth and homeostasis. Deregulation of the pathway has been implicated in many pathological conditions including cancer. Phosphorylation cascades through the pathway have been extensively studied but not much is known about the regulation of gene expression of the pathway components. Here, we report that the mRNA level of eukaryotic translation initiation factor (eIF) subunit 4E-binding protein (4E-BP) gene, one of the key mTOR signaling components, is regulated by the highly conserved Ccr4-Not complex. RNAi knockdown of Not1, a putative scaffold protein of this protein complex, increases the mRNA level of 4E-BP in Drosophila Kc cells. Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3’UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA. These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect. Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression.

Roles of mRNA Fate Modulators Dhh1 and Pat1 in TNRC6-dependent Gene Silencing Recapitulated in Yeast

The CCR4-NOT complex, the major deadenylase in eukaryotes, plays crucial roles in gene expression at the levels of transcription, mRNA decay, and protein degradation. GW182/TNRC6 proteins, which are core components of the microRNA-induced silencing complex in animals, stimulate deadenylation and repress translation via recruitment of the CCR4-NOT complex. Here we report a heterologous experimental system that recapitulates the recruitment of CCR4-NOT complex by TNRC6 in S. cerevisiae. Using this system, we characterize conserved functions of the CCR4-NOT complex. The complex stimulates degradation of mRNA from the 5' end by Xrn1, in a manner independent of both translation and deadenylation. This degradation pathway is probably conserved in miRNA-mediated gene silencing in zebrafish. Furthermore, the mRNA fate modulators Dhh1 and Pat1 redundantly stimulate mRNA decay, but both factors are required for poly(A) tail-independent translation repression by tethered TNRC6A. Our tethering-based reconstitution system reveals that the conserved architecture of Not1/CNOT1 provides a binding surface for TNRC6, thereby connecting microRNA-induced silencing complex to the decapping machinery as well as the translation apparatus.

CCR4-NOT2 Promotes the Differentiation and Lipogenesis of 3T3-L1 Adipocytes via Upregulation of PPARγ, CEBPα and Inhibition of P-GSK3α/β and β-Catenin

Background/Aims: Though CCR4-NOT2 (CNOT2), one of CCR4-NOT complex subunits, was known to be involved in metastasis and apoptosis through transcription and mRNA degradation, its other biological function is poorly understood so far. The aim of this study is to elucidate the molecular role of CNOT2 in the differentiation process of 3T3-L1 preadipocytes. Methods and Results: CNOT2 was overexpressed during the differentiation process of 3T3-L1 preadipocytes. Consistently, mRNA levels of CNOT2, adiponectin, adiponectin 2, PPARγ and CEBPα were enhanced in 3T3-L1 adipocytes. Conversely, CNOT2 depletion by siRNA transfection also reversed the activation of PPARγ and CEBPα and inhibition of GSK3α/β and β-catenin at the protein level in 3T3-L1 preadipocytes. Immunofluorescence assay revealed that CNOT2 was colocalized with PPARγ, but not with CEBPα in 3T3-L1 adipocyte. Consistently, IP western blots revealed that CNOT2 interacted with PPARγ in 3T3-L1 adipocyte. Conclusion: Our findings demonstrate that CNOT2 promotes the differentiation of 3T3-L1 preadipocytes via upregulation of PPARγ, and CEBPα and inhibition of GSK3α/β and β-catenin signaling as a potent molecular target for obesity.

Whole exome sequencing identifies a recurrent RQCD1 P131L mutation in cutaneous melanoma.

Melanoma is often caused by mutations due to exposure to ultraviolet radiation. This study reports a recurrent somatic C > T change causing a P131L mutation in the RQCD1 (Required for Cell Differentiation1 Homolog) gene identified through whole exome sequencing of 20 metastatic melanomas. Screening in 715 additional primary melanomas revealed a prevalence of ~4%. This represents the first reported recurrent mutation in a member of the CCR4-NOT complex in cancer. Compared to tumors without the mutation, the P131L mutant positive tumors were associated with increased thickness (p = 0.02), head and neck (p = 0.009) and upper limb (p = 0.03) location, lentigo maligna melanoma subtype (p = 0.02) and BRAF V600K (p = 0.04) but not V600E or NRAS codon 61 mutations. There was no association with nodal disease (p = 0.3). Mutually exclusive mutations of other members of the CCR4-NOT complex were found in ~20% of the TCGA melanoma dataset suggesting the complex may play an important role in melanoma biology. Mutant RQCD1 was predicted to bind strongly to HLA-A0201 and HLA-Cw3 MHC1 complexes. From thirteen patients with mutant RQCD1, an anti-tumor CD8⁺ T cell response was observed from a single patient's peripheral blood mononuclear cell population stimulated with mutated peptide compared to wildtype indicating a neoantigen may be formed.