共查询到20条相似文献,搜索用时 15 毫秒
1.
GW182 family proteins interact with Argonaute proteins and are required for the translational repression, deadenylation and decay of miRNA targets. To elicit these effects, GW182 proteins interact with poly(A)‐binding protein (PABP) and the CCR4–NOT deadenylase complex. Although the mechanism of miRNA target deadenylation is relatively well understood, how GW182 proteins repress translation is not known. Here, we demonstrate that GW182 proteins decrease the association of eIF4E, eIF4G and PABP with miRNA targets. eIF4E association is restored in cells in which miRNA targets are deadenylated, but decapping is inhibited. In these cells, eIF4G binding is not restored, indicating that eIF4G dissociates as a consequence of deadenylation. In contrast, PABP dissociates from silenced targets in the absence of deadenylation. PABP dissociation requires the interaction of GW182 proteins with the CCR4–NOT complex. Accordingly, NOT1 and POP2 cause dissociation of PABP from bound mRNAs in the absence of deadenylation. Our findings indicate that the recruitment of the CCR4–NOT complex by GW182 proteins releases PABP from the mRNA poly(A) tail, thereby disrupting mRNA circularization and facilitating translational repression and deadenylation. 相似文献
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The GW182 protein colocalizes with mRNA degradation associated proteins hDcp1 and hLSm4 in cytoplasmic GW bodies 总被引:10,自引:2,他引:10
Eystathioy T Jakymiw A Chan EK Séraphin B Cougot N Fritzler MJ 《RNA (New York, N.Y.)》2003,9(10):1171-1173
A novel cytoplasmic compartment referred to as GW bodies (GWBs) was initially identified using antibodies specific to a 182-kD protein termed GW182. GW182 was characterized by multiple glycine(G)-tryptophan(W) repeats and an RNA recognition motif (RRM) that bound a subset of HeLa cell messenger RNAs (mRNAs). The function of GWBs was not known; however, more recent evidence suggested similarities between GWBs and cytoplasmic structures that contain hLSm proteins and hDcp1, the human homolog to a yeast decapping enzyme subunit. In this study, we used antibodies to hLSm4 and hDcp1 to show that both of these markers of an mRNA degradation pathway colocalize to the same structures as GW182. Our studies demonstrate that GW182, hLSm4, and hDcp1 are found in the same cytoplasmic structures and suggest that GW182 is involved in the same mRNA processing pathway as hLSm4 and hDcp1. 相似文献
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GW182 binds to Argonaute (AGO) proteins and has a central role in miRNA‐mediated gene silencing. Using lentiviral shRNA‐induced GW182 knockdown in HEK293 cells, this study identifies a new role of GW182 in regulating miRNA stability. Stably knocking down GW182 or its paralogue TNRC6B reduces transfected miRNA‐mimic half‐lives. Replenishment of GW182 family proteins, as well as one of its domain Δ12, significantly restores the stability of transfected miRNA‐mimic. GW182 knockdown reduces miRNA secretion via secretory exosomes. Targeted siRNA screening identifies a 3′–5′ exoribonuclease complex responsible for the miRNA degradation only when GW182 is knocked down. Immunoprecipitation further confirms that the presence of GW182 in the RISC complex is critical in protecting Argonaute‐bound miRNA. 相似文献
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Ana Eulalio Sigrun Helms Christoph Fritzsch Maria Fauser Elisa Izaurralde 《RNA (New York, N.Y.)》2009,15(6):1067-1077
Proteins of the GW182 family are essential for miRNA-mediated gene silencing in animal cells; they interact with Argonaute proteins (AGOs) and are required for both the translational repression and mRNA degradation mediated by miRNAs. To gain insight into the role of the GW182–AGO1 interaction in silencing, we generated protein mutants that do not interact and tested them in complementation assays. We show that silencing of miRNA targets requires the N-terminal domain of GW182, which interacts with AGO1 through multiple glycine–tryptophan (GW)-repeats. Indeed, a GW182 mutant that does not interact with AGO1 cannot rescue silencing in cells depleted of endogenous GW182. Conversely, silencing is impaired by mutations in AGO1 that strongly reduce the interaction with GW182 but not with miRNAs. We further show that a GW182 mutant that does not localize to P-bodies but interacts with AGO1 rescues silencing in GW182-depleted cells, even though in these cells, AGO1 also fails to localize to P-bodies. Finally, we show that in addition to the N-terminal AGO1-binding domain, the middle and C-terminal regions of GW182 (referred to as the bipartite silencing domain) are essential for silencing. Together our results indicate that miRNA silencing in animal cells is mediated by AGO1 in complex with GW182, and that P-body localization is not required for silencing. 相似文献
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microRNAs (miRNAs) are a conserved class of small RNAs approximately 22 nucleotides in length. They regulate the expression of a large number of mRNAs in animals and plants through the miRNA-induced silencing complex (miRISC). The conserved GW182 family of proteins has recently been identified, and its members have been shown to be associated with miRISC and to be required for miRNA-mediated gene silencing. These proteins have also been localized to processing bodies that are cytoplasmic messenger ribonucleoprotein (mRNP) aggregates containing mRNA decay factors, translational repressors and untranslated mRNAs. Therefore, these properties of GW182 family proteins support the hypothesis that the formation of untranslatable messenger ribonuclear protein particles is one important mechanism of miRNA-mediated gene silencing. 相似文献
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GW182 family proteins are essential for miRNA-mediated gene silencing in animal cells. They are recruited to miRNA targets via interactions with Argonaute proteins and then promote translational repression and degradation of the miRNA targets. The human and Drosophila melanogaster GW182 proteins share a similar domain organization and interact with PABPC1 as well as with subunits of the PAN2-PAN3 and CCR4-NOT deadenylase complexes. The homologous proteins in Caenorhabditis elegans, AIN-1 and AIN-2, lack most of the domains present in the vertebrate and insect proteins, raising the question as to how AIN-1 and AIN-2 contribute to silencing. Here, we show that both AIN-1 and AIN-2 interact with Argonaute proteins through GW repeats in the middle region of the AIN proteins. However, only AIN-1 interacts with C. elegans and D. melanogaster PABPC1, PAN3, NOT1 and NOT2, suggesting that AIN-1 and AIN-2 are functionally distinct. Our findings reveal a surprising evolutionary plasticity of the GW182 protein interaction network and demonstrate that binding to PABPC1 and deadenylase complexes has been maintained throughout evolution, highlighting the significance of these interactions for silencing. 相似文献
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miRNA-mediated gene silencing requires the GW182 proteins, which are characterized by an N-terminal domain that interacts with Argonaute proteins (AGOs), and a C-terminal silencing domain (SD). In Drosophila melanogaster (Dm) GW182 and a human (Hs) orthologue, TNRC6C, the SD was previously shown to interact with the cytoplasmic poly(A)-binding protein (PABPC1). Here, we show that two regions of GW182 proteins interact with PABPC1: the first contains a PABP-interacting motif 2 (PAM2; as shown before for TNRC6C) and the second contains the M2 and C-terminal sequences in the SD. The latter mediates indirect binding to the PABPC1 N-terminal domain. In D. melanogaster cells, the second binding site dominates; however, in HsTNRC6A-C the PAM2 motif is essential for binding to both Hs and DmPABPC1. Accordingly, a single amino acid substitution in the TNRC6A-C PAM2 motif abolishes the interaction with PABPC1. This mutation also impairs TNRC6s silencing activity. Our findings reveal that despite species-specific differences in the relative strength of the PABPC1-binding sites, the interaction between GW182 proteins and PABPC1 is critical for miRNA-mediated silencing in animal cells. 相似文献
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Zhang L Ding L Cheung TH Dong MQ Chen J Sewell AK Liu X Yates JR Han M 《Molecular cell》2007,28(4):598-613
MicroRNAs (miRNAs) regulate gene expression for diverse functions, but only a limited number of mRNA targets have been experimentally identified. We show that GW182 family proteins AIN-1 and AIN-2 act redundantly to regulate the expression of miRNA targets, but not miRNA biogenesis. Immunoprecipitation (IP) and mass spectrometry indicate that AIN-1 and AIN-2 interact only with miRNA-specific Argonaute proteins ALG-1 and ALG-2 and with components of the core translational initiation complex. Known miRNA targets are enriched in AIN-2 complexes, correlating with the expression of corresponding miRNAs. Combining IP with pyrosequencing and microarray analysis of RNAs associated with AIN-1/AIN-2, we identified 106 previously annotated miRNAs plus nine new candidate miRNAs, but nearly no siRNAs, and more than 3500 potential miRNA targets, including nearly all known ones. Our results demonstrate an effective biochemical approach to systematically identify miRNA targets and provide valuable insights regarding the properties of miRNA effector complexes. 相似文献
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Processing bodies (P-bodies) are cytoplasmic foci implicated in the regulation of mRNA translation, storage, and degradation. Key effectors of microRNA (miRNA)-mediated RNA interference (RNAi), such as Argonaute-2 (Ago2), miRNAs, and their cognate mRNAs, are localized to these structures; however, the precise role that P-bodies and their component proteins play in small interfering RNA (siRNA)-mediated RNAi remains unclear. Here, we investigate the relationship between siRNA-mediated RNAi, RNAi machinery proteins, and P-bodies. We show that upon transfection into cells, siRNAs rapidly localize to P-bodies in their native double-stranded conformation, as indicated by fluorescence resonance energy transfer imaging and that Ago2 is at least in part responsible for this siRNA localization pattern, indicating RISC involvement. Furthermore, siRNA transfection induces up-regulated expression of both GW182, a key P-body component, and Ago2, indicating that P-body localization and interaction with GW182 and Ago2 are important in siRNA-mediated RNAi. By virtue of being centers where these proteins and siRNAs aggregate, we propose that the P-body microenvironment, whether as microscopically visible foci or submicroscopic protein complexes, facilitates siRNA processing and siRNA-mediated silencing through the action of its component proteins. 相似文献
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Tadashi Nishihara Latifa Zekri Joerg E. Braun Elisa Izaurralde 《Nucleic acids research》2013,41(18):8692-8705
MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5′-to-3′ messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5′ cap structure by decapping triggers irreversible decay of the mRNA body. Here, we demonstrate that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation. 相似文献
16.
Single‐cell mRNA profiling reveals the hierarchical response of miRNA targets to miRNA induction
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Andrzej J Rzepiela Souvik Ghosh Jeremie Breda Arnau Vina‐Vilaseca Afzal P Syed Andreas J Gruber Katja Eschbach Christian Beisel Erik van Nimwegen Mihaela Zavolan 《Molecular systems biology》2018,14(8)
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FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors
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Ong SH Guy GR Hadari YR Laks S Gotoh N Schlessinger J Lax I 《Molecular and cellular biology》2000,20(3):979-989
The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases. 相似文献
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Identification of distinct cytoplasmic targets for ras/R-ras and rho regulatory proteins 总被引:30,自引:0,他引:30
The protein products of the mammalian ras genes, p21ras, are regulatory guanine nucleotide binding proteins that are involved in the control of cell proliferation, though the exact biochemical processes regulated are unknown. Recently a cytoplasmic protein has been identified that interacts with and increases the GTPase activity of p21ras. It has been shown that this GTPase-activating protein, or GAP, interacts with the effector domain of ras, leading us and others to propose that GAP may be the target for regulation by p21ras. It has become apparent that ras is part of a much larger family of proteins, and at least 15 ras-related genes have now been identified in the mammalian genome. Each encodes a small (about 21 kDa) guanine nucleotide binding protein, but the functions of none of these regulatory molecules are known. We report here that mammalian cytoplasmic extracts contain GAP-like activity toward the products of two other ras-related genes, R-ras and rho. It appears that p23R-ras interacts with the same 125-kDa GAP protein as p21ras whereas p21rho interacts with a distinct 29-kDa protein, rho GAP. 相似文献
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Autophagy represents a signaling-dependent regulated process that allows the degradation of some cellular proteins in autophagosomes, and plays a critical role in the management of cellular homeostasis under various stress conditions. In recent years, selective degradation of cytoplasmic proteins during stress has attracted considerable scientific interest. Here we examined the ability of resveratrol to induce autophagy in a variety of human cancer cell lines. We found that resveratrol-induced autophagy is accompanied by colocalization of proline-, glutamic acid-, and leucine-rich protein-1 (PELP1) with the green fluorescent protein-microtubule-associated protein 1 light chain 3 (GFP-LC3) in autophagosomes. In addition, we found that hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a previously shown PELP1-interacting protein, is co-recruited to autophagosomes in the presence of resveratrol. Although autophagy has been assumed to be a bulk and non-selective degradation process, in recent years, evidence of selective degradation of cytosolic proteins and organelles by autophagy is mounting. These observations suggest that the interaction of the target protein(s) with the delivery protein or proteins such as HRS facilitates the transport of certain cytoplasmic proteins to autophagosomes for their selective degradation, and thus, could influence the cytoplasmic as well as nuclear functions of nuclear receptor coregulators. Since PELP1 and, perhaps, other nuclear receptor coregulators are widely dysregulated in human cancers, these findings highlight the significance of the autophagic selective degradation of PELP1 following resveratrol (or other phytoestrogens) treatment in developing future strategies to use resveratrol under cancer prevention and therapeutic settings. 相似文献