Showing papers in "The EMBO Journal in 2004"
TL;DR: The first direct evidence that miRNA genes are transcribed by RNA polymerase II (pol II) is presented and the detailed structure of a miRNA gene is described, for the first time, by determining the promoter and the terminator of mir‐23a∼27a‐24‐2.
Abstract: MicroRNAs (miRNAs) constitute a large family of noncoding RNAs that function as guide molecules in diverse gene silencing pathways. Current efforts are focused on the regulatory function of miRNAs, while little is known about how these unusual genes themselves are regulated. Here we present the first direct evidence that miRNA genes are transcribed by RNA polymerase II (pol II). The primary miRNA transcripts (pri‐miRNAs) contain cap structures as well as poly(A) tails, which are the unique properties of class II gene transcripts. The treatment of human cells with α‐amanitin decreased the level of pri‐miRNAs at a concentration that selectively inhibits pol II activity. Furthermore, chromatin immunoprecipitation analyses show that pol II is physically associated with a miRNA promoter. We also describe, for the first time, the detailed structure of a miRNA gene by determining the promoter and the terminator of mir‐23a∼27a∼24‐2 . These data indicate that pol II is the main, if not the only, RNA polymerase for miRNA gene transcription. Our study offers a basis for understanding the structure and regulation of miRNA genes.
4,304 citations
TL;DR: It is demonstrated that SIRT1, a nicotinamide adenosine dinucleotide‐dependent histone deacetylase, regulates the transcriptional activity of NF‐κB and activity augments apoptosis in response to TNFα.
Abstract: NF‐κB is responsible for upregulating gene products that control cell survival. In this study, we demonstrate that SIRT1, a nicotinamide adenosine dinucleotide‐dependent histone deacetylase, regulates the transcriptional activity of NF‐κB. SIRT1, the mammalian ortholog of the yeast SIR2 (Silencing Information Regulator) and a member of the Sirtuin family, has been implicated in modulating transcriptional silencing and cell survival. SIRT1 physically interacts with the RelA/p65 subunit of NF‐κB and inhibits transcription by deacetylating RelA/p65 at lysine 310. Treatment of cells with resveratrol, a small‐molecule agonist of Sirtuin activity, potentiates chromatin‐associated SIRT1 protein on the cIAP‐2 promoter region, an effect that correlates with a loss of NF‐κB‐regulated gene expression and sensitization of cells to TNFα‐induced apoptosis. While SIRT1 is capable of protecting cells from p53‐induced apoptosis, our work provides evidence that SIRT1 activity augments apoptosis in response to TNFα by the ability of the deacetylase to inhibit the transactivation potential of the RelA/p65 protein.
2,437 citations
TL;DR: The results show that LKB1 functions as a master upstream protein kinase, regulating AMPK‐related kinases as well as AMPK, and may mediate the physiological effects of L KB1, including its tumour suppressor function.
Abstract: We recently demonstrated that the LKB1 tumour suppressor kinase, in complex with the pseudokinase STRAD and the scaffolding protein MO25, phosphorylates and activates AMP-activated protein kinase (AMPK). A total of 12 human kinases (NUAK1, NUAK2, BRSK1, BRSK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4 and MELK) are related to AMPK. Here we demonstrate that LKB1 can phosphorylate the T-loop of all the members of this subfamily, apart from MELK, increasing their activity >50-fold. LKB1 catalytic activity and the presence of MO25 and STRAD are required for activation. Mutation of the T-loop Thr phosphorylated by LKB1 to Ala prevented activation, while mutation to glutamate produced active forms of many of the AMPK-related kinases. Activities of endogenous NUAK2, QIK, QSK, SIK, MARK1, MARK2/3 and MARK4 were markedly reduced in LKB1-deficient cells. Neither LKB1 activity nor that of AMPK-related kinases was stimulated by phenformin or AICAR, which activate AMPK. Our results show that LKB1 functions as a master upstream protein kinase, regulating AMPK-related kinases as well as AMPK. Between them, these kinases may mediate the physiological effects of LKB1, including its tumour suppressor function.
1,287 citations
TL;DR: Results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.
Abstract: The hormonal action of abscisic acid (ABA) in plants is controlled by the precise balance between its biosynthesis and catabolism. In plants, ABA 8′-hydroxylation is thought to play a predominant role in ABA catabolism. ABA 8′-hydroxylase was shown to be a cytochrome P450 (P450); however, its corresponding gene had not been identified. Through phylogenetic and DNA microarray analyses during seed imbibition, the candidate genes for this enzyme were narrowed down from 272 Arabidopsis P450 genes. These candidate genes were functionally expressed in yeast to reveal that members of the CYP707A family, CYP707A1–CYP707A4, encode ABA 8′-hydroxylases. Expression analyses revealed that CYP707A2 is responsible for the rapid decrease in ABA level during seed imbibition. During drought stress conditions, all CYP707A genes were upregulated, and upon rehydration a significant increase in mRNA level was observed. Consistent with the expression analyses, cyp707a2 mutants exhibited hyperdormancy in seeds and accumulated six-fold greater ABA content than wild type. These results demonstrate that CYP707A family genes play a major regulatory role in controlling the level of ABA in plants.
875 citations
TL;DR: It is shown that mice lacking Suz12 are not viable and die during early postimplantation stages displaying severe developmental and proliferative defects, and an essential role of SUZ12 in regulating the activity of the PRC2/3 complexes, which are required for regulating proliferation and embryogenesis.
Abstract: SUZ12 is a recently identified Polycomb group (PcG) protein, which together with EZH2 and EED forms different Polycomb repressive complexes (PRC2/3). These complexes contain histone H3 lysine (K) 27/9 and histone H1 K26 methyltransferase activity specified by the EZH2 SET domain. Here we show that mice lacking Suz12 , like Ezh2 and Eed mutant mice, are not viable and die during early postimplantation stages displaying severe developmental and proliferative defects. Consistent with this, we demonstrate that SUZ12 is required for proliferation of cells in tissue culture. Furthermore, we demonstrate that SUZ12 is essential for the activity and stability of the PRC2/3 complexes in mouse embryos, in tissue culture cells and in vitro . Strikingly, Suz12‐deficient embryos show a specific loss of di‐ and trimethylated H3K27, demonstrating that Suz12 is indeed essential for EZH2 activity in vivo . In conclusion, our data demonstrate an essential role of SUZ12 in regulating the activity of the PRC2/3 complexes, which are required for regulating proliferation and embryogenesis.
872 citations
TL;DR: A homeostasis mechanism for sustaining cellular reactive oxygen species that is controlled by signalling pathways that can convey both negative (PI‐3K/PKB) and positive (Ras/Ral) inputs is outlined.
Abstract: Forkhead transcription factors of the FOXO class are negatively regulated by PKB/c-Akt in response to insulin/IGF signalling, and are involved in regulating cell cycle progression and cell death. Here we show that, in contrast to insulin signalling, low levels of oxidative stress generated by treatment with H2O2 induce the activation of FOXO4. Upon treatment of cells with H2O2, the small GTPase Ral is activated and this results in a JNK-dependent phosphorylation of FOXO4 on threonine 447 and threonine 451. This Ral-mediated, JNK-dependent phosphorylation is involved in the nuclear translocation and transcriptional activation of FOXO4 after H2O2 treatment. In addition, we show that this signalling pathway is also employed by tumor necrosis factor α to activate FOXO4 transcriptional activity. FOXO members have been implicated in cellular protection against oxidative stress via the transcriptional regulation of manganese superoxide dismutase and catalase gene expression. The results reported here, therefore, outline a homeostasis mechanism for sustaining cellular reactive oxygen species that is controlled by signalling pathways that can convey both negative (PI-3K/PKB) and positive (Ras/Ral) inputs.
745 citations
TL;DR: It is confirmed that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb‐d mutants, supporting a model in which this region of the silencing RNA nucleates pairing to its target.
Abstract: MicroRNAs (miRNAs) are B22-nucleotide noncoding RNAs that can regulate gene expression by directing mRNA degradation or inhibiting productive translation. Dominant mutations in PHABULOSA (PHB )a ndPHAVOLUTA (PHV) map to a miR165/166 complementary site and impair miRNA-guided cleavage of these mRNAs in vitro. Here, we confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb-d mutants. In planta, disrupting miRNA pairing near the center of the miRNA complementary site had far milder developmental consequences than more distal mismatches. These differences correlated with differences in miRNA-directed cleavage efficiency in vitro, where mismatch scanning revealed more tolerance for mismatches at the center and 3 0 end of the miRNA compared to mismatches to the miRNA 5 0 region. In this respect, miR165/ 166 resembles animal miRNAs in its pairing requirements. Pairing to the 5 0 portion of the small silencing RNA appears crucial regardless of the mode of post-transcriptional repression or whether it occurs in plants or animals, supporting a model in which this region of the silencing RNA nucleates pairing to its target.
714 citations
TL;DR: It is shown that the F‐box protein Fbw7 interacts with and thereby destabilizes c‐Myc in a manner dependent on phosphorylation of MB1, suggesting that two F‐ box proteins, FbW7 and Skp2, differentially regulate c‐ myc stability by targeting MB1 and MB2, respectively.
Abstract: The F-box protein Skp2 mediates c-Myc ubiquitylation by binding to the MB2 domain. However, the turnover of c-Myc is largely dependent on phosphorylation of threonine-58 and serine-62 in MB1, residues that are often mutated in cancer. We now show that the F-box protein Fbw7 interacts with and thereby destabilizes c-Myc in a manner dependent on phosphorylation of MB1. Whereas wild-type Fbw7 promoted c-Myc turnover in cells, an Fbw7 mutant lacking the F-box domain delayed it. Furthermore, depletion of Fbw7 by RNA interference increased both the abundance and transactivation activity of c-Myc. Accumulation of c-Myc was also apparent in mouse Fbw7−/− embryonic stem cells. These observations suggest that two F-box proteins, Fbw7 and Skp2, differentially regulate c-Myc stability by targeting MB1 and MB2, respectively.
709 citations
TL;DR: It is reported that expression of the hairy/enhancer‐of‐split‐related transcriptional repressor Hey1, and the Notch‐ligand Jagged1 (Jag1), was induced by TGF‐β at the onset of EMT in epithelial cells from mammary gland, kidney tubules, and epidermis.
Abstract: Epithelial‐to‐mesenchymal transitions (EMTs) underlie cell plasticity required in embryonic development and frequently observed in advanced carcinogenesis. Transforming growth factor‐β (TGF‐β) induces EMT phenotypes in epithelial cells in vitro and has been associated with EMT in vivo . Here we report that expression of the hairy/enhancer‐of‐split‐related transcriptional repressor Hey1, and the Notch‐ligand Jagged1 (Jag1), was induced by TGF‐β at the onset of EMT in epithelial cells from mammary gland, kidney tubules, and epidermis. The HEY1 expression profile was biphasic, consisting of immediate‐early Smad3‐dependent, Jagged1/Notch‐independent activation, followed by delayed, indirect Jagged1/Notch‐dependent activation. TGF‐β‐induced EMT was blocked by RNA silencing of HEY1 or JAG1 , and by chemical inactivation of Notch. The EMT phenotype, biphasic activation of Hey1 , and delayed expression of Jag1 were induced by TGF‐β in wild‐type, but not in Smad3‐deficient, primary mouse kidney tubular epithelial cells. Our findings identify a new mechanism for functional integration of Jagged1/Notch signalling and coordinated activation of the Hey1 transcriptional repressor controlled by TGF‐β/Smad3, and demonstrate functional roles for Smad3, Hey1, and Jagged1/Notch in mediating TGF‐β‐induced EMT.
681 citations
TL;DR: The results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation and blocks TGF‐β‐induced growth arrest by indirectly reducing T GF‐β/ALK5 signalling.
Abstract: Endoglin is a transmembrane accessory receptor for transforming growth factor-β (TGF-β) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-β signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-β type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-β/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-β/ALK1 signalling, which indirectly inhibits TGF-β/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-β/ALK1 signalling is reduced and TGF-β/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-β-induced growth arrest by indirectly reducing TGF-β/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.
634 citations
TL;DR: It is shown that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity.
Abstract: Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, after apoptosis induction, translocates to the nucleus where it participates in apoptotic chromatinolysis. Here, we show that human or mouse cells lacking AIF as a result of homologous recombination or small interfering RNA exhibit high lactate production and enhanced dependency on glycolytic ATP generation, due to severe reduction of respiratory chain complex I activity. Although AIF itself is not a part of complex I, AIF-deficient cells exhibit a reduced content of complex I and of its components, pointing to a role of AIF in the biogenesis and/or maintenance of this polyprotein complex. Harlequin mice with reduced AIF expression due to a retroviral insertion into the AIF gene also manifest a reduced oxidative phosphorylation (OXPHOS) in the retina and in the brain, correlating with reduced expression of complex I subunits, retinal degeneration, and neuronal defects. Altogether, these data point to a role of AIF in OXPHOS and emphasize the dual role of AIF in life and death.
TL;DR: It is demonstrated that HIF‐1α induces cell cycle arrest by functionally counteracting Myc, thereby derepressing p21cip1, and proposed that Myc is an integral part of a novel HIF-1α pathway, which regulates a distinct group of Myc target genes in response to hypoxia.
Abstract: Hypoxia induces angiogenesis and glycolysis for cell growth and survival, and also leads to growth arrest and apoptosis. HIF-1α, a basic helix–loop–helix PAS transcription factor, acts as a master regulator of oxygen homeostasis by upregulating various genes under low oxygen tension. Although genetic studies have indicated the requirement of HIF-1α for hypoxia-induced growth arrest and activation of p21cip1, a key cyclin-dependent kinase inhibitor controlling cell cycle checkpoint, the mechanism underlying p21cip1 activation has been elusive. Here we demonstrate that HIF-1α, even in the absence of hypoxic signal, induces cell cycle arrest by functionally counteracting Myc, thereby derepressing p21cip1. The HIF-1α antagonism is mediated by displacing Myc binding from p21cip1 promoter. Neither HIF-1α transcriptional activity nor its DNA binding is essential for cell cycle arrest, indicating a divergent role for HIF-1α. In keeping with its antagonism of Myc, HIF-1α also downregulates Myc-activated genes such as hTERT and BRCA1. Hence, we propose that Myc is an integral part of a novel HIF-1α pathway, which regulates a distinct group of Myc target genes in response to hypoxia.
TL;DR: The major receptors responsible for NK cell triggering are NKp46, NK p30, NKp44 and NKG2D as discussed by the authors, which are specific for major histocompatibility complex (MHC) class I molecules, allowing NK cells to discriminate between normal cells and cells that have lost the expression of MHC class I (e.g., tumor cells).
Abstract: Natural killer (NK) cells represent a highly specialized lymphoid population characterized by a potent cytolytic activity against tumor or virally infected cells. Their function is finely regulated by a series of inhibitory or activating receptors. The inhibitory receptors, specific for major histocompatibility complex (MHC) class I molecules, allow NK cells to discriminate between normal cells and cells that have lost the expression of MHC class I (e.g., tumor cells). The major receptors responsible for NK cell triggering are NKp46, NKp30, NKp44 and NKG2D. The NK-mediated lysis of tumor cells involves several such receptors, while killing of dendritic cells involves only NKp30. The target-cell ligands recognized by some receptors have been identified, but those to which major receptors bind are not yet known. Nevertheless, functional data suggest that they are primarily expressed on cells upon activation, proliferation or tumor transformation. Thus, the ability of NK cells to lyse target cells requires both the lack of surface MHC class I molecules and the expression of appropriate ligands that trigger NK receptors.
TL;DR: The structure of the ectodomain of the tick‐borne encephalitis virus envelope glycoprotein, E, a prototypical class II fusion protein, in its trimeric low‐pH‐induced conformation is reported, suggesting an important conformational effect of the missing membrane connecting segment.
Abstract: Enveloped viruses enter cells via a membrane fusion reaction driven by conformational changes of specific viral envelope proteins. We report here the structure of the ectodomain of the tick-borne encephalitis virus envelope glycoprotein, E, a prototypical class II fusion protein, in its trimeric low-pH-induced conformation. We show that, in the conformational transition, the three domains of the neutral-pH form are maintained but their relative orientation is altered. Similar to the postfusion class I proteins, the subunits rearrange such that the fusion peptide loops cluster at one end of an elongated molecule and the C-terminal segments, connecting to the viral transmembrane region, run along the sides of the trimer pointing toward the fusion peptide loops. Comparison with the low-pH-induced form of the alphavirus class II fusion protein reveals striking differences at the end of the molecule bearing the fusion peptides, suggesting an important conformational effect of the missing membrane connecting segment.
TL;DR: It is shown that polη does not form nuclear foci in RAD18−/− cells after UV irradiation, and Rad18 is crucial for recruitment ofPolη to the damaged site through protein–protein interaction and PCNA monoubiquitination.
Abstract: The DNA replication machinery stalls at damaged sites on templates, but normally restarts by switching to a specialized DNA polymerase(s) that carries out translesion DNA synthesis (TLS). In human cells, DNA polymerase η (polη) accumulates at stalling sites as nuclear foci, and is involved in ultraviolet (UV)-induced TLS. Here we show that polη does not form nuclear foci in RAD18−/− cells after UV irradiation. Both Rad18 and Rad6 are required for polη focus formation. In wild-type cells, UV irradiation induces relocalization of Rad18 in the nucleus, thereby stimulating colocalization with proliferating cell nuclear antigen (PCNA), and Rad18/Rad6-dependent PCNA monoubiquitination. Purified Rad18 and Rad6B monoubiquitinate PCNA in vitro. Rad18 associates with polη constitutively through domains on their C-terminal regions, and this complex accumulates at the foci after UV irradiation. Furthermore, polη interacts preferentially with monoubiquitinated PCNA, but polδ does not. These results suggest that Rad18 is crucial for recruitment of polη to the damaged site through protein–protein interaction and PCNA monoubiquitination.
TL;DR: It is demonstrated that activated JNK promotes Bax translocation to mitochondria through phosphorylation of 14‐3‐3, a cytoplasmic anchor of Bax, a key mechanism of BAX regulation in stress‐induced apoptosis.
Abstract: Targeted gene disruption studies have established that the c-Jun NH2-terminal kinase (JNK) is required for the stress-induced release of mitochondrial cytochrome c and apoptosis, and that the Bax subfamily of Bcl-2-related proteins is essential for JNK-dependent apoptosis. However, the mechanism by which JNK regulates Bax has remained unsolved. Here we demonstrate that activated JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3, a cytoplasmic anchor of Bax. Phosphorylation of 14-3-3 led to dissociation of Bax from this protein. Expression of phosphorylation-defective mutants of 14-3-3 blocked JNK-induced Bax translocation to mitochondria, cytochrome c release and apoptosis. Collectively, these results have revealed a key mechanism of Bax regulation in stress-induced apoptosis.
TL;DR: It now appears that one needs to Take Five genes to produce a deadly peptide by a proteolytic mechanism, which paradoxically is otherwise of pivotal importance for development and cell fate decisions.
Abstract: In 1959, Dave Brubeck and Paul Desmond revolutionized modern jazz music by composing their unforgettable Take Five in 5/4, one of the most defiant time signatures in all music. Of similar revolutionary importance for biomedical and basic biochemical research is the identification of the minimal set of genes required to obtain a deadly time bomb ticking in all of us: Alzheimer's disease. It now appears that one needs to Take Five genes to produce a deadly peptide by a proteolytic mechanism, which paradoxically is otherwise of pivotal importance for development and cell fate decisions.
TL;DR: Using a functional cloning strategy in yeast, a novel family of integral membrane proteins exhibiting all enzymatic features previously attributed to animal SM synthase is identified, and these findings open up important new avenues for studying sphingolipid function in animals.
Abstract: Sphingomyelin (SM) is a major component of animal plasma membranes. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product. This reaction is catalysed by SM synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and ceramide as anti- and proapoptotic stimuli, respectively. SM synthesis occurs in the lumen of the Golgi as well as on the cell surface. As no gene for SM synthase has been cloned so far, it is unclear whether different enzymes are present at these locations. Using a functional cloning strategy in yeast, we identified a novel family of integral membrane proteins exhibiting all enzymatic features previously attributed to animal SM synthase. Strikingly, human, mouse and Caenorhabditis elegans genomes each contain at least two different SM synthase (SMS) genes. Whereas human SMS1 is localised to the Golgi, SMS2 resides primarily at the plasma membrane. Collectively, these findings open up important new avenues for studying sphingolipid function in animals.
TL;DR: The most recent advances in the field of E2F transcription factors are summarised and how specificity is achieved among the E2Fs is focused on.
Abstract: The E2F transcription factors are key regulators of cell cycle progression and the E2F field has made rapid advances since its advent in 1986. Yet, while our understanding of the roles and functions of the E2F family has made enormous progress, with each discovery new questions arise. In this review, we summarise the most recent advances in the field and discuss the remaining key questions. In particular, we will focus on how specificity is achieved among the E2Fs.
TL;DR: It is shown that muscles of juvenile Pax7(−/−) mice at P11 contain a reduced but substantial number of satellite cells, indicating an essential function of Pax7 for renewal and maintenance of muscle stem cells and exclude an exclusive role of Pax8 in satellite cell specification.
Abstract: The paired-box transcription factor Pax7 has been claimed to specify the muscle stem cell lineage since inactivation of Pax7 led to a failure to detect muscle satellite cells. Here we show that muscles of juvenile Pax7(−/−) mice at P11 contain a reduced but substantial number of satellite cells. Neither juvenile nor adult Pax7(−/−) mice displayed a significant reduction in the number and size of myotubes, indicating that the remaining number of satellite cells sufficed to allow normal postnatal muscle growth. The number of satellite cells in Pax7 mutant mice declined strongly during postnatal development, although single satellite cells were readily identified in adult Pax7 mutant mice. Muscle regeneration was impaired in adult Pax7 mutant mice. Our results clearly indicate an essential function of Pax7 for renewal and maintenance of muscle stem cells and exclude an exclusive role of Pax7 in satellite cell specification.
TL;DR: The data reveal the mechanism by which the p38‐MAPK/MK2 kinase cascade inhibits TTP‐mediated degradation of ARE‐containing transcripts and thereby contributes to lipopolysaccharide‐induced TNFα expression.
Abstract: Stress granules (SGs) are dynamic cytoplasmic foci at which stalled translation initiation complexes accumulate in cells subjected to environmental stress. SG‐associated proteins such as TIA‐1, TIAR and HuR bind to AU‐rich element (ARE)‐containing mRNAs and control their translation and stability. Here we show that tristetraprolin (TTP), an ARE‐binding protein that destabilizes ARE‐mRNAs, is recruited to SGs that are assembled in response to FCCP‐induced energy deprivation, but not arsenite‐induced oxidative stress. Exclusion of TTP from arsenite‐induced SGs is a consequence of MAPKAP kinase‐2 (MK2)‐induced phosphorylation at serines 52 and 178, which promotes the assembly of TTP:14‐3‐3 complexes. 14‐3‐3 binding excludes TTP from SGs and inhibits TTP‐dependent degradation of ARE‐containing transcripts. In activated RAW 264.7 macrophages, endogenous TTP:14‐3‐3 complexes bind to ARE‐RNA. Our data reveal the mechanism by which the p38‐MAPK/MK2 kinase cascade inhibits TTP‐mediated degradation of ARE‐containing transcripts and thereby contributes to lipopolysaccharide‐induced TNFα expression.
TL;DR: It is argued that HIV‐1 Vif has evolved to suppress at least two distinct but related human antiretroviral DNA‐editing enzymes.
Abstract: The HIV-1 Vif protein suppresses the inhibition of viral replication caused by the human antiretroviral factor APOBEC3G. As a result, HIV-1 mutants that do not express the Vif protein are replication incompetent in 'nonpermissive' cells, such as primary T cells and the T-cell line CEM, that express APOBEC3G. In contrast, Vif-defective HIV-1 replicates effectively in 'permissive' cell lines, such as a derivative of CEM termed CEM-SS, that do not express APOBEC3G. Here, we show that a second human protein, APOBEC3F, is also specifically packaged into HIV-1 virions and inhibits their infectivity. APOBEC3F binds the HIV-1 Vif protein specifically and Vif suppresses both the inhibition of virus infectivity caused by APOBEC3F and virion incorporation of APOBEC3F. Surprisingly, APOBEC3F and APOBEC3G are extensively coexpressed in nonpermissive human cells, including primary lymphocytes and the cell line CEM, where they form heterodimers. In contrast, both genes are quiescent in the permissive CEM derivative CEM-SS. Together, these data argue that HIV-1 Vif has evolved to suppress at least two distinct but related human antiretroviral DNA-editing enzymes.
TL;DR: The results indicate that the composition and fate of HuR‐ and/or AUF1‐containing ribonucleoprotein complexes depend on the target mRNA of interest, RNA‐binding protein abundance, stress condition, and subcellular compartment.
Abstract: RNA-binding proteins HuR and AUF1 bind to many common AU-rich target mRNAs and exert opposing influence on target mRNA stability, but the functional interactions between HuR and AUF1 have not been systematically studied. Here, using common target RNAs encoding p21 and cyclin D1, we provide evidence that HuR and AUF1 can bind target transcripts on both distinct, nonoverlapping sites, and on common sites in a competitive fashion. In the nucleus, both proteins were found together within stable ribonucleoprotein complexes; in the cytoplasm, HuR and AUF1 were found to bind to target mRNAs individually, HuR colocalizing with the translational apparatus and AUF1 with the exosome. Our results indicate that the composition and fate (stability, translation) of HuR- and/or AUF1-containing ribonucleoprotein complexes depend on the target mRNA of interest, RNA-binding protein abundance, stress condition, and subcellular compartment.
TL;DR: TREK-1 is a two-pore-domain background potassium channel expressed throughout the central nervous system and has been suggested to be an important target in the action of these drugs as discussed by the authors.
Abstract: TREK-1 is a two-pore-domain background potassium channel expressed throughout the central nervous system. It is opened by polyunsaturated fatty acids and lysophospholipids. It is inhibited by neurotransmitters that produce an increase in intracellular cAMP and by those that activate the Gq protein pathway. TREK-1 is also activated by volatile anesthetics and has been suggested to be an important target in the action of these drugs. Using mice with a disrupted TREK-1 gene, we now show that TREK-1 has an important role in neuroprotection against epilepsy and brain and spinal chord ischemia. Trek1−/− mice display an increased sensitivity to ischemia and epilepsy. Neuroprotection by polyunsaturated fatty acids, which is impressive in Trek1+/+ mice, disappears in Trek1−/− mice indicating a central role of TREK-1 in this process. Trek1−/− mice are also resistant to anesthesia by volatile anesthetics. TREK-1 emerges as a potential innovative target for developing new therapeutic agents for neurology and anesthesiology.
TL;DR: In this article, the authors provide biochemical and structural evidence that macro domains are high affinity binding modules for ADP-ribose nucleotide nucleotides and reveal a conserved ligand binding pocket among the macro domain fold.
Abstract: The ADP-ribosylation of proteins is an important post-translational modification that occurs in a variety of biological processes, including DNA repair, transcription, chromatin biology and long-term memory formation. Yet no protein modules are known that specifically recognize the ADP-ribose nucleotide. We provide biochemical and structural evidence that macro domains are high-affinity ADP-ribose binding modules. Our structural analysis reveals a conserved ligand binding pocket among the macro domain fold. Consistently, distinct human macro domains retain their ability to bind ADP-ribose. In addition, some macro domain proteins also recognize poly-ADP-ribose as a ligand. Our data suggest an important role for proteins containing macro domains in the biology of ADP-ribose.
TL;DR: It is proposed that eIF4B may mediate some of the effects of the S6Ks on translation, and specifically phosphorylation of Ser422, located in an RNA‐binding region required for eif4A helicase‐promoting activity.
Abstract: The eucaryotic translation initiation factor 4B (eIF4B) stimulates the helicase activity of the DEAD box protein eIF4A to unwind inhibitory secondary structure in the 5′ untranslated region of eucaryotic mRNAs. Here, using phosphopeptide mapping and a phosphospecific antiserum, we identify a serum-responsive eIF4B phosphorylation site, Ser422, located in an RNA-binding region required for eIF4A helicase-promoting activity. Ser422 phosphorylation appears to be regulated by the S6Ks: (a) Ser422 phosphorylation is sensitive to pharmacological inhibitors of phosphoinositide-3 kinase and the mammalian target of rapamycin; (b) S6K1/S6K2 specifically phosphorylate Ser422 in vitro; and (c) rapamycin-resistant S6Ks confer rapamycin resistance upon Ser422 phosphorylation in vivo. Substitution of Ser422 with Ala results in a loss of activity in an in vivo translation assay, indicating that phosphorylation of this site plays an important role in eIF4B function. We therefore propose that eIF4B may mediate some of the effects of the S6Ks on translation.
TL;DR: It is demonstrated that MBNL proteins regulate alternative splicing of two pre‐mRNAs that are misregulated in DM, cardiac troponin T (cTNT) and insulin receptor (IR).
Abstract: Although the muscleblind (MBNL) protein family has been implicated in myotonic dystrophy (DM), a specific function for these proteins has not been reported. A key feature of the RNA-mediated pathogenesis model for DM is the disrupted splicing of specific pre-mRNA targets. Here we demonstrate that MBNL proteins regulate alternative splicing of two pre-mRNAs that are misregulated in DM, cardiac troponin T (cTNT) and insulin receptor (IR). Alternative cTNT and IR exons are also regulated by CELF proteins, which were previously implicated in DM pathogenesis. MBNL proteins promote opposite splicing patterns for cTNT and IR alternative exons, both of which are antagonized by CELF proteins. CELF- and MBNL-binding sites are distinct and regulation by MBNL does not require the CELF-binding site. The results are consistent with a mechanism for DM pathogenesis in which expanded repeats cause a loss of MBNL and/or gain of CELF activities, leading to misregulation of alternative splicing of specific pre-mRNA targets.
TL;DR: The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes.
Abstract: Mitochondria import nuclear-encoded precursor proteins to four different subcompartments. Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively. However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far. We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w). Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10. The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40. The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes. We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.
TL;DR: It is reported that collagenases bind and locally unwind the triple‐helical structure before hydrolyzing the peptide bonds and shows that MMP‐1 preferentially interacts with the α2(I) chain of type I collagen and cleaves the three α chains in succession.
Abstract: Breakdown of triple-helical interstitial collagens is essential in embryonic development, organ morphogenesis and tissue remodelling and repair. Aberrant collagenolysis may result in diseases such as arthritis, cancer, atherosclerosis, aneurysm and fibrosis. In vertebrates, it is initiated by collagenases belonging to the matrix metalloproteinase (MMP) family. The three-dimensional structure of a prototypic collagenase, MMP-1, indicates that the substrate-binding site of the enzyme is too narrow to accommodate triple-helical collagen. Here we report that collagenases bind and locally unwind the triple-helical structure before hydrolyzing the peptide bonds. Mutation of the catalytically essential residue Glu200 of MMP-1 to Ala resulted in a catalytically inactive enzyme, but in its presence noncollagenolytic proteinases digested collagen into typical 3/4 and 1/4 fragments, indicating that the MMP-1(E200A) mutant unwinds the triple-helical collagen. The study also shows that MMP-1 preferentially interacts with the α2(I) chain of type I collagen and cleaves the three α chains in succession. Our results throw light on the basic mechanisms that control a wide range of biological and pathological processes associated with tissue remodelling.
TL;DR: It is reported, using TIS21‐GFP knock‐in mouse embryos to identify neurogenic neuroepithelial cells, that at the onset as well as advanced stages of neurogenesis the vast majority of neurogenic divisions, like proliferative divisions, show vertical cleavage planes.
Abstract: At the onset of neurogenesis in the mammalian central nervous system, neuroepithelial cells switch from symmetric, proliferative to asymmetric, neurogenic divisions. In analogy to the asymmetric division of Drosophila neuroblasts, this switch of mammalian neuroepithelial cells is thought to involve a change in cleavage plane orientation from perpendicular (vertical cleavage) to parallel (horizontal cleavage) relative to the apical surface of the neuroepithelium. Here, we report, using TIS21-GFP knock-in mouse embryos to identify neurogenic neuroepithelial cells, that at the onset as well as advanced stages of neurogenesis the vast majority of neurogenic divisions, like proliferative divisions, show vertical cleavage planes. Remarkably, however, neurogenic divisions of neuroepithelial cells, but not proliferative ones, involve an asymmetric distribution to the daughter cells of the apical plasma membrane, which constitutes only a minute fraction (1–2%) of the entire neuroepithelial cell plasma membrane. Our results support a novel concept for the cell biological basis of asymmetric, neurogenic divisions of neuroepithelial cells in the mammalian central nervous system.