A distinct mammalian disome collision interface harbors K63-linked polyubiquitination of uS10 to trigger hRQT-mediated subunit dissociation.

Translational stalling events that result in ribosome collisions induce Ribosome-associated Quality Control (RQC) in order to degrade potentially toxic truncated nascent proteins. For RQC induction, the collided ribosomes are first marked by the Hel2/ZNF598 E3 ubiquitin ligase to recruit the RQT complex for subunit dissociation. In yeast, uS10 is polyubiquitinated by Hel2, whereas eS10 is preferentially monoubiquitinated by ZNF598 in human cells for an unknown reason. Here, we characterize the ubiquitination activity of ZNF598 and its importance for human RQT-mediated subunit dissociation using the endogenous XBP1u and poly(A) translation stallers. Cryo-EM analysis of a human collided disome reveals a distinct composite interface, with substantial differences to yeast collided disomes. Biochemical analysis of collided ribosomes shows that ZNF598 forms K63-linked polyubiquitin chains on uS10, which are decisive for mammalian RQC initiation. The human RQT (hRQT) complex composed only of ASCC3, ASCC2 and TRIP4 dissociates collided ribosomes dependent on the ATPase activity of ASCC3 and the ubiquitin-binding capacity of ASCC2. The hRQT-mediated subunit dissociation requires the K63-linked polyubiquitination of uS10, while monoubiquitination of eS10 or uS10 is not sufficient. Therefore, we conclude that ZNF598 functionally marks collided mammalian ribosomes by K63-linked polyubiquitination of uS10 for the trimeric hRQT complex-mediated subunit dissociation.

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.

Regulation of Fetal Genes by Transitions among RNA-Binding Proteins during Liver Development.

Transcripts of alpha-fetoprotein (Afp), H19, and insulin-like growth factor 2 (Igf2) genes are highly expressed in mouse fetal liver, but decrease drastically during maturation. While transcriptional regulation of these genes has been well studied, the post-transcriptional regulation of their developmental decrease is poorly understood. Here, we show that shortening of poly(A) tails and subsequent RNA decay are largely responsible for the postnatal decrease of Afp, H19, and Igf2 transcripts in mouse liver. IGF2 mRNA binding protein 1 (IMP1), which regulates stability and translation efficiency of target mRNAs, binds to these fetal liver transcripts. When IMP1 is exogenously expressed in mouse adult liver, fetal liver transcripts show higher expression and possess longer poly(A) tails, suggesting that IMP1 stabilizes them. IMP1 declines concomitantly with fetal liver transcripts as liver matures. Instead, RNA-binding proteins (RBPs) that promote RNA decay, such as cold shock domain containing protein E1 (CSDE1), K-homology domain splicing regulatory protein (KSRP), and CUG-BP1 and ETR3-like factors 1 (CELF1), bind to 3' regions of fetal liver transcripts. These data suggest that transitions among RBPs associated with fetal liver transcripts shift regulation from stabilization to decay, leading to a postnatal decrease in those fetal transcripts.

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 γ (PPARγ). Collectively, these findings suggest that paracrine signaling related to liver function influences hair growth, at least in part, by altering lipid metabolism.

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 Krüppel-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 Krüppel-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 Krüppel-like factor 2 and p21, contrary to tumor suppressive functions of CNOT3 in T-ALL.

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.

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.

Stability of mRNA influences osteoporotic bone mass via CNOT3.

Osteoclastogenesis is under the control of posttranscriptional and transcriptional events. However, posttranscriptional regulation of osteoclastogenesis is incompletely understood. CNOT3 is a component of the CCR4 family that regulates mRNA stability, but its function in bone is not known. Here, we show that Cnot3 deficiency by deletion of a single allele induces osteoporosis. Cnot3 deficiency causes an enhancement in bone resorption in association with an elevation in bone formation, resulting in high-turnover type bone loss. At the cellular level, Cnot3 deficiency enhances receptor activator of NF-κB ligand (RANKL) effects on osteoclastogenesis in a cell-autonomous manner. Conversely, Cnot3 deficiency does not affect osteoblasts directly. Cnot3 deficiency does not alter RANKL expression but enhances receptor activator of NF-κB (RANK) mRNA expression in bone in vivo. Cnot3 deficiency promotes RANK mRNA stability about twofold in bone marrow cells of mice. Cnot3 knockdown also increases RANK mRNA expression in the precursor cell line for osteoclasts. Anti-CNOT3 antibody immunoprecipitates RANK mRNA. Cnot3 deficiency stabilizes luciferase reporter expression linked to the 3'-UTR fragment of RANK mRNA. In contrast, Cnot3 overexpression destabilizes the luciferase reporter linked to RANK 3'-UTR. In aged mice that exhibit severe osteoporosis, Cnot3 expression levels in bone are reduced about threefold in vivo. Surprisingly, Cnot3 deficiency in these aged mice further exacerbates osteoporosis, which also occurs via enhancement of osteoclastic activity. Our results reveal that CNOT3 is a critical regulator of bone mass acting on bone resorption through posttranscriptional down-regulation of RANK mRNA stability, at least in part, even in aging-induced osteoporosis.

Ubiquitin-specific protease 2-69 in macrophages potentially modulates metainflammation.

Macrophages play a critical role in chronic inflammation and metabolic diseases. We identified a longer splice variant of ubiquitin specific protease (USP) 2-69 as a novel molecule that modulates pathways implicated in metabolic disorders. Expression levels of aP2/FABP4 and PAI-1/SERPINE1 genes were increased by 4- and 1.8-fold, respectively, after short hairpin RNA-mediated knockdown (KD) of the USP2 gene, and such expression was alleviated by overexpression of USP2-69 in human myeloid cell lines. Supernatants derived from USP2-KD cells induced IL6 (∼6-fold) and SAA3 (∼15-fold) in 3T3-L1 adipocytes to suggest the anti-inflammatory properties of USP2. In addition, we observed a 30% decrease in the number of macrophages in mesenteric adipose tissue derived from USP2-69 transgenic mice fed a high-fat diet for 14 wk compared with that in their C57BL/6 littermates (P<0.01), which was consistent with a ∼40% decrease in transcription of aP2 and PAI-1. The aP2 locus exhibited elevated chromatin accessibility (>2.1-fold), methylation of histone H3 lysine 4 (>4.5-fold), and acetylation of histone H4 (>2.5-fold) in USP2-KD cells. Transfection of isopeptidase-mutated USP2-69 did not alter chromatin conformation on the aP2 locus in USP2-KD cells. Our results suggest that USP2-69 suppresses meta-inflammatory molecules involved in the development of type-2 diabetes.

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.

SP600125 inhibits cap-dependent translation independently of the c-Jun N-terminal kinase pathway.

We investigated the effects of SP600125 (formerly called c-Jun N-terminal kinase (JNK) inhibitor II) on translation using cultured mouse cells. SP600125 (50 µM) treatment rapidly repressed overall protein synthesis, accompanied by a reduction in the mRNAs for housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase in the polysomal fraction. SP600125 decreased polysomes with a concomitant increase in free ribosomal subunits in the cytoplasm, suggesting that global translation was inhibited at the initiation step. A reporter analysis using exogenous mRNAs showed that SP600125 inhibited cap-dependent but not internal ribosome entry site-dependent translation. SP600125 significantly attenuated phosphorylation of components in the mTOR pathway, which is responsible for cap-dependent translation. In contrast to SP600125, short hairpin RNAs for JNK1 and JNK2 failed to affect overall protein synthesis. Collectively, SP600125 inhibits cap-dependent translation, independent of the JNK pathway.

Genome-wide identification and characterization of transcripts translationally regulated by bacterial lipopolysaccharide in macrophage-like J774.1 cells.

Although Escherichia coli LPS is known to elicit various proinflammatory responses in macrophages, its effect on the translational states of transcripts has not yet been explored on a genome-wide scale. To address this, we investigated the mRNA profiles in polysomal and free messenger ribonucleoprotein particle (mRNP) fractions of mouse macrophage-like J774.1 cells, using Affymetrix Mouse Genome 430 2.0 GeneChips. Comparison of the mRNA profiles in total cellular, polysomal, and free mRNP fractions enabled us to identify transcripts that were modulated at the translational level by LPS: among 19,791 transcripts, 115 and 418 were up- and downregulated at 1, 2, or 4 h after LPS stimulation (100 ng/ml) in a translation-dependent manner. Interestingly, gene ontology-based analysis suggested that translation-dependent downregulated genes frequently include those encoding proteins in the mitochondrial respiratory chain. In fact, the mRNA levels of some transcripts for complexes I, IV, and V in the mitochondrial respiratory chain were translationally downregulated, eventually contributing to the decline of their protein levels. Moreover, the amount of metabolically labeled cytochrome oxidase subunit Va in complex IV was decreased without any change of its mRNA level in total cellular fraction after LPS stimulation. Consistently, the total amounts and activities of complexes I and IV were attenuated by LPS stimulation, and the attenuation was independent of nitric oxide. These results demonstrated that translational suppression may play a critical role in the LPS-mediated attenuation of mitochondrial oxidative phosphorylation in a nitric oxide-independent manner in J774.1 cells.