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Development of lymphocytes is precisely regulated by various mechanisms. In addition to transcriptional rates, post-transcriptional regulation of mRNA abundance contributes to differentiation of lymphocytes. mRNA decay is a post-transcriptional mechanism controlling mRNA abundance. The carbon catabolite repression 4 (CCR4)-negative on TATA-less (NOT) complex controls mRNA longevity by catalyzing mRNA deadenylation, which is the rate-limiting step in the mRNA decay pathway. mRNA decay, regulated by the CCR4-NOT complex, is required for differentiation of pro-B to pre-B cells and V(D)J recombination in pro-B cells. In this process, it is likely that the RNA-binding proteins, ZFP36 ring finger protein like 1 and 2, recruit the CCR4-NOT complex to specific target mRNAs, thereby inducing cell quiescence of pro-B cells. A recent study showed that the CCR4-NOT complex participates in positive selection of thymocytes. Mechanistically, the CCR4-NOT deadenylase complex inhibits abnormal apoptosis by reducing the expression level of mRNAs encoding pro-apoptotic proteins, which are otherwise up-regulated during positive selection. We discuss mechanisms regulating CCR4-NOT complex-dependent mRNA decay in lymphocyte development and selection. [ABSTRACT FROM AUTHOR]

Molecular left-right (L-R) asymmetry is established at the node of the mouse embryo as a result of the sensing of a leftward fluid flow by immotile cilia of perinodal crown cells and the consequent degradation of Dand5 mRNA on the left side. We here examined how the fluid flow induces Dand5 mRNA decay. We found that the first 200 nucleotides in the 3′ untranslated region (3′-UTR) of Dand5 mRNA are necessary and sufficient for the left-sided decay and to mediate the response of a 3′-UTR reporter transgene to Ca2+, the cation channel Pkd2, the RNA-binding protein Bicc1 and their regulation by the flow direction. We show that Bicc1 preferentially recognizes GACR and YGAC sequences, which can explain the specific binding to a conserved GACGUGAC motif located in the proximal Dand5 3′-UTR. The Cnot3 component of the Ccr4-Not deadenylase complex interacts with Bicc1 and is also required for Dand5 mRNA decay at the node. These results suggest that Ca2+ currents induced by leftward fluid flow stimulate Bicc1 and Ccr4-Not to mediate Dand5 mRNA degradation specifically on the left side of the node. Questioning what regulates left-right asymmetry breaking in the mouse node: the authors identify a 200 bp stretch of the Dand5 3'UTR where Bicc1 binds, and Cnot proteins downstream of calcium flow regulate the post-transcriptional regulation of Dand5 by Bicc1. [ABSTRACT FROM AUTHOR]

Although ATP-sensitive K+ (KATP) channels play an important role in the secretion of insulin by pancreatic beta cells, the mechanisms that regulate the intracellular transport of KATP channel subunit proteins (i.e., Kir6.2 and sulfonylurea receptor 1 (SUR1)) to the plasma membrane remain uncharacterized. We investigated the possibility that an interaction between KATP channel subunit proteins and Rab8a protein, a member of the RAS superfamily, may be involved in the membrane trafficking of KATP channels. Co-immunoprecipitation and immunostaining experiments using co-expression systems with fluorescent protein-tagged Kir6.2 were carried out to identify the coupling of KATP channels and Rab8a proteins in the insulin-secreting cell line, MIN6. Rab8a protein co-localized with Kir6.2 protein, a channel pore subunit (in a granular pattern), and with insulin. Knockdown of the Rab8a gene with RNA interference using small interfering RNA systems caused reductions in the amount of total KATP and plasma membrane surface KATP channels without decreasing the messenger RNA transcription of the KATP channel subunits. Rab8a gene knockdown also enhanced glucose-induced insulin secretion. These results suggest that Rab8a may be involved in membrane trafficking of KATP channels and the maintenance of normal insulin secretion in the MIN6 pancreatic beta cell line. [ABSTRACT FROM AUTHOR]

In mammals, γ-tubulin, a key protein in microtubule nucleation, is encoded by two genes, TUBG1 and TUBG2. Human TUBG1 and TUBG2 mRNA are expressed ubiquitously and predominantly in preimplantation embryos and the brain, respectively, but specific detection of γ-tubulin2 protein expression is difficult due to their high sequence similarity. Here, we describe a protocol for differential detection of two human γ-tubulins by western blotting. In several cancer cell lines and the brain, expression of γ-tubulin2 along with γ-tubulin1 and a novel TUBG2 splice variant are identified. Contribution of ectopically expressed γ-tubulin2 in cancer growth was determined by depletion of γ-tubulin2. [ABSTRACT FROM AUTHOR]

Obesity is a life-threatening factor and is often associated with dysregulation of gene expression. Here, we show that the CNOT3 subunit of the CCR4-NOT deadenylase complex is critical to metabolic regulation. Cnot3+/? mice are lean with hepatic and adipose tissues containing reduced levels of lipids, and show increased metabolic rates and enhanced glucose tolerance. Cnot3+/? mice remain lean and sensitive to insulin even on a high-fat diet. Furthermore, introduction of Cnot3 haplodeficiency in ob/ob mice ameliorated the obese phenotype. Hepatic expression of most mRNAs is not altered in Cnot3+/? vis-à-vis wild-type mice. However, the levels of specific mRNAs, such as those coding for energy metabolism-related PDK4 and IGFBP1, are increased in Cnot3+/? hepatocytes, having poly(A) tails that are longer than those seen in control cells. We provide evidence that CNOT3 is involved in recruitment of the CCR4-NOT deadenylase to the 3? end of specific mRNAs. Finally, as CNOT3 levels in the liver and white adipose tissues decrease upon fasting, we propose that CNOT3 responds to feeding conditions to regulate deadenylation-specific mRNAs and energy metabolism. [ABSTRACT FROM AUTHOR]

miRNAs recruit the miRNA-induced silencing complex (miRISC), which includes Argonaute and GW182 as core proteins. GW182 proteins effect translational repression and deadenylation of target mRNAs. However, the molecular mechanisms of GW182-mediated repression remain obscure. We show here that human GW182 independently interacts with the PAN2-PAN3 and CCR4-NOT deadenylase complexes. Interaction of GW182 with CCR4-NOT is mediated by two newly discovered phylogenetically conserved motifs. Although either motif is sufficient to bind CCR4-NOT, only one of them can promote processive deadenylation of target mRNAs. Thus, GW182 serves as both a platform that recruits deadenylases and as a deadenylase coactivator that facilitates the removal of the poly(A) tail by CCR4-NOT. [ABSTRACT FROM AUTHOR]

Eukaryotic mRNA decay is initiated by shortening of the poly (A) tail; however, neither the molecular mechanisms underlying deadenylation nor its regulation is well understood. The human CCR4-NOT complex is a major cytoplasmic deadenylase consisting of a combination of at least nine subunits, four of which have deadenylase activity. The roles of the other subunits remain obscure. Here, we show that CNOT2 depletion by siRNA induces apoptosis. We also show that CNOT2 depletion destabilizes the complex, resulting in the formation of a complex smaller than that formed in control siRNA-treated cells. The deadenylase activity of the CNOT6L subunit-containing complex prepared from CNOT2-depleted cells was less than that from control cells. Intriguingly, the formation of P-bodies, where mRNA degradation supposedly takes place, was largely suppressed in CNOT2-depleted cells. Furthermore, CNOT2 depletion enhanced CHOP mRNA levels, suggesting that endoplasmic reticulum (ER) stress was occurring, which causes apoptosis in a caspase-dependent manner. These results suggest that CNOT2 is important for controlling cell viability through the maintenance of the structural integrity and enzymatic activity of the CCR4-NOT complex. [ABSTRACT FROM AUTHOR]

Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under nephrotic conditions. Their requisite components, claudins, still remain to be identified. We have measured the mRNA levels of claudin subtypes by quantitative real-time PCR using isolated rat glomeruli. Claudin-5 was found to be expressed most abundantly in glomeruli. Mass spectrometric analysis of membrane preparation from isolated glomeruli also confirmed only claudin-5 expression without any detection of other claudin subtypes. In situ hybridization and immunolocalization studies revealed that claudin-5 was localized mainly in glomeruli where podocytes were the only cells expressing claudin-5. Claudin-5 protein was observed on the entire surface of podocytes including apical and basal domains of the plasma membrane in the normal condition and was inclined to be concentrated on tight junctions in puromycin aminonucleoside nephrosis. Total protein levels of claudin-5 in isolated glomeruli were not significantly upregulated in the nephrosis. These findings suggest that claudin-5 is a main claudin expressed in podocytes and that the formation of tight junctions in the nephrosis may be due to local recruitment of claudin-5 rather than due to total upregulation of the claudin protein levels. [ABSTRACT FROM AUTHOR]

Avian kidneys have loopless and looped nephrons; a countercurrent multiplier mechanism operates in the latter by NaCl recycling. We identified an aquaporin-2 (AQP2) homolog in apical/subapical regions of cortical and medullary collecting duct (CD) cells in kidneys of Japanese quail (q), Coturnix japonica. We investigated whether undernutrition during the embryonic/maturation period retards kidney and AQP2 development in quail and programs impaired volume regulation in adults. Protocols included 1) time course and 2) effects of 5-10% egg white withdrawal (EwW) or 48-h post-hatch food deprivation (FD) on nephron growth and qAQP2 mRNA expression, and 3) effects of EwW and FD on qAQP2 mRNA responses to 72-h water deprivation in adults. In metanephric kidneys, qAQP2 mRNA is expressed in medullary CDs at embryonic day 10; distribution and intensity increase during maturation. The number and size of glomeruli continue to increase after birth, whereas nephrogenic zones decrease. In EwW embryos, qAQP2 mRNA expression is initially delayed, then restored; birth weight and hatching rate are lower than in controls. Adults from EwW embryos and FD chicks have fewer (P < 0.01) glomeruli. Water deprivation reduces body weight more in EwW birds than in controls. The results suggest that qAQP2 evolved in metanephric kidneys and that undernutrition may retard nephrogenesis, leading to impaired adult water homeostasis. [ABSTRACT FROM AUTHOR]

Neurotensin receptor subtype 2 (Ntsr2) is a levocabastine-sensitive neurotensin receptor expressed diffusely throughout the mouse brain. Previously, we found that Ntsr2-deficient mice have an abnormality in the processing of thermal nociception. In this study, to examine the involvement of Ntsr2 in mouse behavior, we performed a fear-conditioning test in Ntsr2-deficient mice. In the contextual fear-conditioning test, the freezing response was significantly reduced in Ntsr2-deficient mice compared with that of wild-type mice. This reduction was observed from 1 h to 3 weeks after conditioning, and neither shock sensitivity nor locomotor activity was altered in Ntsr2-deficient mice. In addition, we found that Ntsr2 mRNA was predominantly expressed in cultured astrocytes and weakly expressed in cultured neurons derived from mouse brain. The combination of in situ hybridization and immunohistochemistry showed that Ntsr2 mRNA was dominantly expressed in glial fibrillary acidic protein positive cells in many brain regions including the hypothalamus, while Ntsr2 gene was co-expressed with neuron-specific microtubule associated protein-2 in limited numbers of cells. These results suggest that Ntsr2 in astrocytes and neurons may have unique function like a modulation of fear memory in the mouse brain. [ABSTRACT FROM AUTHOR]

Hagfish (Epmtretus burgeri) are agnathous and are the earliest vertebrates still in existence. Pavement cells adjacent to the mitochondria-rich cells show orthogonal arrays of particles (OAPs) in the gill of hagfish, a known ultrastructural morphology of aquaporin (AQP) in mammalian freeze-replica studies, suggesting that an AQP homolog exists in pavement cells. We therefore cloned water channels from hagfish gill and examined their molecular characteristics. The cloned AQP [E. burgeri AQP4 (EbAQP4)] encodes 288 amino acids, including two NPA motifs and six transmembrane regions. The deduced amino acid sequence of EbAQP4 showed high homology to mammalian and avian AQP4 (rat, 44%; quail, 43%) and clustered with AQP4 subsets by the molecular phylogenetic tree. The osmotic water permeability of Xenopus oocytes injected with EbAQP4 cRNA increased eightfold compared with water-injected controls and was not reversibly inhibited by 0.3 mM HgCl2. EbAQP4 mRNA expression in the gill was demonstrated by the RNase protection assay; antibody raised against the COOH terminus of EbAQP4 also detected (by Western blot analysis) a major ∼31-kDa band in the gill. Immunohistochemistry and immunoelectron microscopy showed EbAQP4 localized along the basolateral membranes of gill pavement cells. In freeze-replica studies, OAPs were detected on the protoplasmic face of the split membrane comprising particles 5–6 nm long on the basolateral side of the pavement cells. These observations suggest that EbAQP4 is an ancestral water channel of mammalian AQP4 and plays a role in basolateral water transport in the gill pavement cells. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) regulate cellular fate by controlling the stability or translation of mRNA transcripts. Although the spatial and temporal patterning of miRNA expression is tightly controlled, little is known about signals that induce their expression nor mechanisms of their transcriptional regulation. Furthermore, few miRNA targets have been validated experimentally. The miRNA, miR132, was identified through a genome-wide screen as a target of the transcription factor, cAMP-response element binding protein (CREB). miR132 is enriched in neurons and, like many neuronal CREB targets, is highly induced by neurotrophins. Expression of miR132 in cortical neurons induced neurite outgrowth. Conversely, inhibition of miR132 function attenuated neuronal outgrowth. We provide evidence that miR132 regulates neuronal morphogenesis by decreasing levels of the GTPase-activating protein, p250GAP. These data reveal that a CREB-regulated miRNA regulates neuronal morphogenesis by responding to extrinsic trophic cues. [ABSTRACT FROM AUTHOR]

Tob is a member of an emerging family of anti-proliferative proteins that suppress cell growth when over-expressed.tobmRNA is highly expressed in anergic T cells and over-expression of Tob suppresses transcription of interleukin-2 (IL-2) through its interaction with Smads. Here, we identified two types of cDNA clones coding for poly(A)-binding protein (PABP) and inducible PABP (iPABP) by screening an expression cDNA library with the GST-Tob probe. Co-immunoprecipitation and GST-pull down experiments showed that Tob associated with the carboxyl-terminal region of iPABP. We then found that iPABP, like PABP, was involved in regulation of translation: iPABP enhanced translation of IL-2 mRNAin vitro. The enhanced translation of IL-2 mRNA required the 3′UTR and poly(A) sequences. Tob abrogated the enhancement of translation through its interaction with carboxyl-terminal region of iPABPin vitro. Consistently, over-expression of Tob in NIH3T3 cells, in which exogenous iPABP was stably expressed, resulted in suppression of IL-2 production from the simultaneously transfected IL-2 expression plasmid. Finally, Tob, whose expression was induced by anergic stimulation, was co-immunoprecipitated with iPABP in human T cells. These findings suggest that Tob is involved in the translational suppression of IL-2 mRNA in anergic T cells through its interaction with iPABP. [ABSTRACT FROM AUTHOR]

Background. Matrix metalloproteinase (MMP)-9, a member of the MMP family with specificity towards type IV collagen, is implicated in the turnover of the extracellular matrix in the kidney. To elucidate its physiological and pathophysiological significance, we examined the expression and localization of MMP-9 in the normal kidney and the changes in these features during the course of antiglomerular basement membrane (GBM) glomerulonephritis induced in WKY rats, along with the changes in these features of tissue inhibitor of metalloproteinase 1 (TIMP-1) and MMP-2. Methods. The expression of MMP-9, TIMP-1 and MMP-2 mRNA was quantified by ribonuclease protection assay, and the gelatinolytic activities of MMP-9 and MMP-2 were evaluated by gelatin zymography. The localization of MMP-9 was visualized by immunohistochemistry and immunofluorescence microscopy. Results. The ribonuclease protection assay indicated the almost exclusive expression of MMP-9 mRNA in the glomerulus of normal kidneys. Immunohistochemistry and double-label immunofluorescence microscopy showed that MMP-9 was localized in the mesangial cells. During the course of anti-GBM glomerulonephritis, the expression of MMP-9 mRNA in glomeruli increased on day 1, peaked on days 3 to 7, and then decreased on day 14. The change in MMP-9 mRNA expression was accompanied by parallel changes in the gelatinolytic activity of the active form of MMP-9, TIMP-1 mRNA expression, and MMP-9 immunoreactivity in mesangial cells. In contrast, glomerular MMP-2 mRNA expression and its activity increased after the decline of MMP-9. Conclusions. MMP-9 mRNA was predominantly expressed in the glomerulus in normal rat kidneys and MMP-9 was present in the mesangium. The MMP-9 mRNA expression increased in the glomerulus 3 to 7 days after the induction of anti-GBM glomerulonephritis in WKY rats, in parallel with the development of abnormal glomerular histology and injury, suggesting a role of MMP-9 in proteolysis of the GBM during glomerulonephritis. MMP-2 may participate in the later phase of the nephritis. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study is to isolate mechanical stress-induced genes (MSGens) from human periodontal ligament (PDL) cells and to analyze profiles of the mRNA expression of these genes. Background: Differential expression of genes in PDL cells under physiological stress such as occlusal force is thought to be orchestrated not only for the remodeling of PDL itself but also for the repair and regeneration of periodontal tissues. However, little is known about the genes expressed in PDL cells under mechanical stress. Methods: The cDNA from mechanical stress-applied human PDL cells was subtracted against the cDNA from static control cells. The subtracted cDNA was amplified by polymerase chain reaction (PCR) and cloned for further analysis. Results: Among 68 independent clones isolated, 15 contained DNA fragments greater than 250 bp. Reverse Northern analysis revealed a marked induction of MSGen-15 and MSGen-28 mRNA expression in the mechanical stress-applied cells. However, little difference in the magnitude of expression for the other MSGens was detected between the stress-applied cells and the control cells. After nucleotide sequencing and the analysis of homology with known genes, five clones were identified; ribosomal protein S27 (MSGen-9), MRG 15 (MSGen-15), androgen-binding protein (MSGen-18), cathepsin H (MSGen-28), and cytochrome c (MSGen-47). Interestingly, it has been reported that MRG 15 is a novel transcription factor involved in the regulation of cell growth and senescence. The remaining 10 clones, classified into six sequence types, had no significant homology with any known genes. Conclusions: These results suggest that many known and unknown genes are expressed in response to mechanical stress in PDL cells, and that a transcription factor, MRG 15, may be responsible for molecular events in PDL cells under mechanical stress. [ABSTRACT FROM AUTHOR]

Examines the expression and distribution of several types of aquaporins in the gastrointestinal tract in rats. Quantitative evaluation of aquaporin family mRNA expression in rat gastrointestinal tract; Possible role of water channels in water absorption or secretion in the gastrointestinal tract.

Examines the messenger ribonucleic acid (mRNA) expression of the aquaporin (AQP) family. Localization of AQP2 and AQP3 during the development of the rat kidneys; Staining intensity of the AQP proteins; Contribution to the maturation of urinary concentrating capacity.

Examines the expression of messenger RNA for natriuretic peptide receptor subtypes in bovine kidney. Quantitative analysis of the ribonuclease protection assay; In situ hybridization; Regulation of glomerular filtration rate and sodium excretion.

Expression of mRNA for IL-1α, IL-1β, IL-2. IL-4, IL-5, IL-6 and TNF-α in inflamed gingiva was quantitatively examined by ribonuclease protection assay and in situ hybridization. The IL-1β mRNA expression level was statistically high (P<0.05) in periodontitis-affected tissues compared with that in gingivitis-affected tissues. The densities of macrophages (identified as CD68-positive cells) and CD45RO-positive cells infiltrating in the inflamed gingiva correlated statistically with IL-1β transcript levels (macrophages, P<0.001; CD45RO-positive cells. P<0.002). In situ hybridization revealed IL-β mRNA expression in infiltrating cells, presumed to be macrophages. The IL-1α and IL-6 mRNA expression levels were much lower than the IL-1β transcript level, and mRNAs for IL-2, IL-4, IL-5 and TNF-α were negligible in these gingival tissues. The results indicate that IL-1β is a cytokine expressed predominantly in inflamed gingiva and reflects the density of infiltrating macrophages and other leukocytes. [ABSTRACT FROM AUTHOR]

The exact cell type and site(s) involved in interleukin-1 (IL-1) production during gingival inflammation was determined by combining immunohistochemistry and in situ hybridization. IL-1 messenger RNA (mRNA)-expressing cells in human inflamed gingiva were identified as macrophages. The rat of IL-α mRNA expression in these macrophages was the same as IL-1β mRNA expression. The rate of IL-1 mRNA expression was higher in connective tissue furthest from the pocket epithelium, although more macrophages were present at the connective tissue subjacent to the pocket epithelium. The IL-1 activity in gingival crevicular fluid (GCF) obtained from inflamed gingiva was higher than that from healthy gingiva and decreased after periodontal therapy. The IL-1 activity in GCF was almost completely abolished by the addition of anti-IL-1α antibody but not by anti-IL-1β antibody, indicating that IL-1α is the predominant form in GCF. However, the IL-1 activity in GCF was unrelated to the number of IL-1 mRNA-expressing macrophages in the same gingival site where the GCF was obtained at the same time. The results suggest that macrophages in the connective tissue subjacent to the oral epithelium contribute to the production of IL-1 but those in connective tissue subjacent to the pocket epithelium play a different role in the generation of gingival inflammation. [ABSTRACT FROM AUTHOR]

The 2;5 chromosomal translocation is frequently associated with anaplastic large cell lymphomas (ALCLs). The translocation creates a fusion gene consisting of the alk (anaplastic lymphoma kinase) gene and the nucelophosmin (npm) gene: the 3′ half of alk derived from chromosome 2 is fused to the 5′ portion of npm from chromosome 5. A recent study shows that the product of the npm-alk fusion gene is oncogenic. To help understand how the npm-alk oncogene transform cells, it is important to investigate the normal biological function of the alk gene product, ALK. Here, we show molecular cloning of cDNAs for both the human and mouse ALK proteins. The deduced amino acid sequences reveal that ALK is a novel receptor protein-tyrosine kinase having a putative transmembrane domain and an extracellular domain. These sequences are absent in the product of the transforming npm-alk gene. ALK shows the greatest sequence similarity to LTK (leukocyte tyrosine kinase) whose biological function is presently unknown. RNA blot hybridization analysis of various tissues reveals that the alk mRNA is dominantly detected in the brain and spinal cord. Immunoblotting with anti-ALK antibody shows that ALK is highly expressed in the neonatal brain. Furthermore, RNA in situ hybridization analysis shows that the alk mRNA is dominantly expressed in neurons in specific regions of the nervous system such as the thalamus, mid-brain, olfactory bulb, and ganglia of embryonic and neonatal mice. These data suggest that ALK plays an important role(s) in the development of the brain and exerts its effects on specific neurons in the nervous system. [ABSTRACT FROM AUTHOR]

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. The CCR4-NOT complex catalyzes mRNA deadenylation and hence regulates protein translation. Here the authors show that CNOT3 regulation of this complex is needed for positive selection of thymocytes via a mechanism involving inhibition of pro-apoptotic gene expression. [ABSTRACT FROM AUTHOR]

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. Dina Mostafa et al. report that β cell function and identity depends on the deadenylase complex subunit CNOT3. The authors found that Cnot3 was dysregulated in diabetic mice and, when knocked out in wild type, mice have impaired glucose tolerance and gradually develop diabetes. [ABSTRACT FROM AUTHOR]

A correction to the article "MiRNA-Mediated Deadenylation is Orchestrated by GW182 Through Two Conserved Motifs That Interact With CCR4-NOT" in the November 8, 2011 issue is presented.

The stability of mRNA influences the abundance of cellular transcripts and proteins. Deadenylases play critical roles in mRNA turnover and thus are important for the regulation of various biological events. Here, we report the identification and characterization of CCR4b/CNOT6L, which is homologous to yeast CCR4 mRNA deadenylase. CCR4b is localized mainly in the cytoplasm and displays deadenylase activity both in vitro and in vivo. CCR4b forms a multisubunit complex similar to the yeast CCR4-NOT complex. Suppression of CCR4b by RNA interference results in growth retardation of NIH 3T3 cells accompanied by elevation of both p27Kip1 mRNA and p27Kip1 protein. Reintroduction of wild-type CCR4b, but not mutant CCR4b lacking deadenylase activity, restores the growth of CCR4b-depleted NIH 3T3 cells. The data suggest that CCR4b regulates cell growth in a manner dependent on its deadenylase activity. We also show that p27Kip1 mRNA is stabilized and its poly(A) tail is preserved in CCR4b-depleted cells. Our findings provide evidence that CCR4b deadenylase is a constituent of the mammalian CCR4-NOT complex and regulates the turnover rate of specific target mRNAs. Thus, CCR4b may be involved in various cellular events that include cell proliferation. [ABSTRACT FROM AUTHOR]

[Display omitted] • Functional differences between CCR4-NOT subunits CNOT7 and CNOT8 have been elusive. • Despite high similarity, CNOT8 protein was significantly less stable than CNOT7. • Stability of CNOT8, but not CNOT7, was increased by its interaction with CNOT1. • High-affinity binding of CNOT7 to CNOT1 excluded CNOT8 from the CCR4-NOT complex. • CNOT7 is likely to be the dominant paralog in 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. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies found that the NMDA receptor-mediated signaling regulates thermal nociception, though the underlying molecular mechanism remains unclear. The GluN2B subunit of the NMDA receptor is tyrosine-phosphorylated, Tyr-1472 being the major phosphorylation site. In this study, we have found that homozygous knock-in mice that express a Tyr-1472-Phe mutant of GluN2B display defects in the nociceptive response in the hot plate test. Expression of the neurotensin receptor subtype 2 (NTSR2), which is relevant to the regulation of thermal nociception, is decreased in the amygdala of GluN2B Tyr-1472-Phe knock-in mice. In addition, NTSR2-mediated c-fos induction is impaired in the amygdala of these mice. These data suggest that Tyr-1472 phosphorylation on GluN2B is involved in thermal nociception through regulating the NTSR2 mRNA expression in the amygdala. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The GTPase-activating proteins for Rho family GTPases (RhoGAP) transduce diverse intracellular signals by negatively regulating Rho family GTPase-mediated pathways. In this study, we have cloned and characterized a novel RhoGAP for Rac1 and Cdc42, termed RRC-1, from Caenorhabditis elegans. RRC-1 was highly homologous to mammalian p250GAP and promoted GTP hydrolysis of Rac1 and Cdc42 in cells. The rrc-1 mRNA was expressed in all life stages. Using an RRC-1::GFP fusion protein, we found that RRC-1 was localized to the coelomocytes, excretory cell, GLR cells, and uterine-seam cell in adult worms. These data contribute toward understanding the roles of Rho family GTPases in C. elegans. [Copyright &y& Elsevier]

Fyn, a member of the Src family of tyrosine kinases, is implicated in both brain development and adult brain function. In the present study, we identified a Rho GTPase-activating protein (GAP), TCGAP (Tc10/Cdc42 GTPase-activating protein), as a novel Fyn substrate. TCGAP interacted with Fyn and was phosphorylated by Fyn, with Tyr-406 in the GAP domain as a major Fyn-mediated phosphorylation site. Fyn suppressed the GAP activity of wild-type TCGAP but not the Y406F mutant of TCGAP in a phosphorylation-dependent manner, suggesting that Fyn-mediated Tyr-406 phosphorylation negatively regulated the TCGAP activity. In situ hybridization analyses showed that TCGAP mRNA was expressed prominently in both immature and adult mouse brain, with high levels in cortex, corpus striatum, hippocampus, and olfactory bulb. Overexpression of wild-type TCGAP in PC12 cells suppressed nerve growth factor-induced neurite outgrowth, whereas a GAP-defective mutant of TCGAP enhanced the neurite outgrowth. Nerve growth factor enhanced tyrosine phosphorylation of TCGAP through activation of Src family kinases. These results suggest that TCGAP is involved in Fyn-mediated regulation of axon and dendrite outgrowth. [ABSTRACT FROM AUTHOR]

Tob is a member of the Tob/BTG family, a novel class of anti-proliferative proteins. To investigate the involvement of tob as a tumor suppressor gene in human lung cancer, we analyzed the expression of tob mRNA and protein in lung cancer tissue and adjacent normal lung tissue. Immunohistochemical analysis using anti-Tob antibody showed decreased expression of Tob in 72% (31/43) of lung cancer tissues. Furthermore, 95% (19/20) of squamous cell carcinoma patients showed an apparent decrease in Tob in cancer tissues, associated with smoking status. The phosphorylated form of Tob, an inactive form of Tob, was detected in 76% (16/21) of cancer tissues of adenocarcinoma patients, but not in normal alveolar epithelial cells. Either a decrease in Tob expression or an accumulation of phosphorylated Tob was observed from early clinical stages, even in bronchial dysplasia, a premalignant lesion of squamous cell carcinoma. The above findings suggest that the disruption of anti-proliferative Tob plays a distinct part in the early stage of lung carcinogenesis. [Copyright &y& Elsevier]

• Synthetic (+)-terrein suppressed RANKL-induced osteoclast differentiation and function. • The regulation by synthetic (+)-terrein may be mediated through RANKL-NFATc1 axis. • As an osteoclast-targeting small molecule inhibitor, (+)-terrein shows potential as therapeutic drugs. Pathophysiological bone resorption is commonly associated with periodontal disease and involves the excessive resorption of bone matrix by activated osteoclasts. Receptor activator of nuclear factor (NF)-κB ligand (RANKL) signaling pathways have been proposed as targets for inhibiting osteoclast differentiation and bone resorption. The fungal secondary metabolite (+)-terrein is a natural compound derived from Aspergillus terreus that has previously shown anti-interleukin-6 properties related to inflammatory bone resorption. However, its effects and molecular mechanism of action on osteoclastogenesis and bone resorption remain unclear. In the present study, we showed that 10 µM synthetic (+)-terrein inhibited RANKL-induced osteoclast formation and bone resorption in a dose-dependent manner and without cytotoxicity. RANKL-induced messenger RNA expression of osteoclast-specific markers including nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) , the master regulator of osteoclastogenesis, cathepsin K , tartrate-resistant acid phosphatase (Trap) was completely inhibited by synthetic (+)-terrein treatment. Furthermore, synthetic (+)-terrein decreased RANKL-induced NFATc1 protein expression. This study revealed that synthetic (+)-terrein attenuated osteoclast formation and bone resorption by mediating RANKL signaling pathways, especially NFATc1, and indicated the potential effect of (+)-terrein on inflammatory bone resorption including periodontal disease. [ABSTRACT FROM AUTHOR]