Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures

Dijk, Erwin van; Cougot, Nicolas; Meyer, Sylke; Babajko, Sylvie; Wahle, Elmar; Séraphin, Bertrand

doi:10.1093/emboj/cdf678

Cited by 427 publications

(465 citation statements)

References 37 publications

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“…GWB are named from the marker protein GW182, which contains multiple glycine (G) and tryptophan (W) repeats and a classic RNA binding domain at the carboxyl terminus (Eystathioy et al, 2002a). The mRNA decay factors/complexes found in GWB include the deadenylase Ccr4, the decapping complex Dcp1a/1b/Dcp2, the LSm1-7 complex, Ge-1 (also known as Hedls), rck/p54, and exonuclease Xrn1 (Bashkirov et al, 1997;van Dijk et al, 2002;Ingelfinger et al, 2002;Lykke-Andersen, 2002;Eystathioy et al, 2003;Cougot et al, 2004;Andrei et al, 2005;Yu et al, 2005;Fenger-Gron et al, 2005). GWB are physically juxtaposed to and transiently interact with stress granules (SG).…”

Section: Introductionmentioning

confidence: 99%

Small Interfering RNA-mediated Silencing Induces Target-dependent Assembly of GW/P Bodies

Lian¹,

Fritzler

Katz³

et al. 2007

MBoC

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Gene silencing using small interfering RNA (siRNA) is a valuable laboratory tool and a promising approach to therapeutics for a variety of human diseases. Recently, RNA interference (RNAi) has been linked to cytoplasmic GW bodies (GWB). However, the correlation between RNAi and the formation of GWB, also known as mammalian processing bodies, remains unclear. In this report, we show that transfection of functional siRNA induced larger and greater numbers of GWB. This siRNA-induced increase of GWB depended on the endogenous expression of the target mRNA. Knockdown of GW182 or Ago2 demonstrated that the siRNA-induced increase of GWB required these two proteins and correlated with RNAi. Furthermore, knockdown of rck/p54 or LSm1 did not prevent the reassembly of GWB that were induced by and correlated with siRNA-mediated RNA silencing. We propose that RNAi is a key regulatory mechanism for the assembly of GWB, and in some cases, GWB may serve as markers for RNAi in mammalian cells. INTRODUCTIONGW bodies (GWB), also known as mammalian processing bodies (P bodies), are cytoplasmic foci that contain multiple decay factors and that are involved in the 5Ј33Ј mRNA degradation pathway. GWB are named from the marker protein GW182, which contains multiple glycine (G) and tryptophan (W) repeats and a classic RNA binding domain at the carboxyl terminus (Eystathioy et al., 2002a). The mRNA decay factors/complexes found in GWB include the deadenylase Ccr4, the decapping complex Dcp1a/1b/Dcp2, the LSm1-7 complex, Ge-1 (also known as Hedls), rck/p54, and exonuclease Xrn1 (Bashkirov et al., 1997;van Dijk et al., 2002;Ingelfinger et al., 2002;Lykke-Andersen, 2002;Eystathioy et al., 2003;Cougot et al., 2004;Andrei et al., 2005;Yu et al., 2005;Fenger-Gron et al., 2005). GWB are physically juxtaposed to and transiently interact with stress granules (SG). SG process cytoplasmic aggregates of stalled translational preinitiation complexes that accumulate during stress responses and that share certain components with GWB (Kedersha et al., 2005).In addition to mRNA decay, a crucial role of GWB and their components in RNA interference (RNAi) was recently uncovered (Anderson and Kedersha, 2006;Eulalio et al., 2007a;Jakymiw et al., 2007). RNAi is a posttranscriptional gene silencing mechanism that uses specific doublestranded RNA to silence genes in a sequence-specific manner Mello and Conte, 2004). In brief, the double-stranded RNA is processed by Dicer into small interfering RNA (siRNA) or microRNA (miRNA). The 21-to 26-nucleotide siRNA and miRNA are then incorporated in the effector complex, RNA-induced silencing complex (RISC), which either cleaves or inhibits translation of the target mRNA. In 2005, two key components of RISC, Argonaute2 (Ago2) and siRNA/miRNA, were found to be enriched in GWB (Sen and Blau, 2005;Pillai et al., 2005;Jakymiw et al., 2005;Liu et al., 2005b;Pauley et al., 2006). miRNA-targeted mRNA also localizes to GWB in a miRNAdependent manner (Liu et al., 2005b). These observations provide the first evidence that RNAi is ...

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Section: Introductionmentioning

confidence: 99%

Small Interfering RNA-mediated Silencing Induces Target-dependent Assembly of GW/P Bodies

Lian¹,

Fritzler

Katz³

et al. 2007

MBoC

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“…DNA Constructs. pNEGFP-hDCP1a (van Dijk et al, 2002;Pillai et al, 2005) was kind gift from Dr. Jens Lykke-Andersen (University of Colorado, Boulder, CO), transfected as 0.4 g/well (24-well plate).…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

“…Quantification of siLuc and siSMN colocalization to P-bodies showed a lower but again highly significant colocalization coefficient (siLuc, 0.159 Ϯ 0.09; siSMN, 0.15 Ϯ 0.03, p Ͻ 0.01). On monitoring siRNA localization in live cells expressing the P-body decapping enzyme component hDcp1a tagged with eGFP (eGFP-hDcp1a) to visualize P-bodies (van Dijk et al, 2002;Pillai et al, 2005), we saw that siRNAs could be found in P-bodies within 30 min of transfection ( Supplemental Figure 1). Some large bodies containing intense siRNA signals were also present; these have been described previously to be vesicles/endosomes that remained from transfection (Jakymiw et al, 2005).…”

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confidence: 99%

Localization of Double-stranded Small Interfering RNA to Cytoplasmic Processing Bodies Is Ago2 Dependent and Results in Up-Regulation of GW182 and Argonaute-2

Jagannath

Wood

2009

MBoC

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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. INTRODUCTIONRNA interference (RNAi) is a powerful homology-based gene silencing mechanism directed by small RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs). RNAi is a posttranscriptional process, leading to either translational repression of the target mRNA, typical of miRNAmediated silencing, or degradation of the target via siRNAmediated silencing (Hammond, 2005). Recently, several components of the RNAi pathway, including Argonaute proteins (Sen and Blau, 2005), miRNAs, and their targets (Liu et al., 2005b;Pillai et al., 2005) have been localized to processing bodies (P-bodies). P-bodies are recognized as important cytoplasmic mRNA processing centers where nontranslating mRNA is sorted and either stored, repressed, or degraded (for review, see Eulalio et al., 2007a). Enzymes associated with mRNA degradation, such the CCR4-CAF-1-Not complex involved in deadenlyation (Cougot et al., 2004); DCP1 and -2 (van Dijk et al., 2002) involved in decapping; and XRN-1, a 5Ј-3Ј exonuclease (Ingelfinger et al., 2002), have all been localized to P-bodies. These foci also contain proteins involved in nonsense-mediated decay (Sheth and Parker, 2006) and AU-rich element-mediated mRNA decay pathways (Vasudevan and Steitz, 2007).Although it is clear that RNAi and P-bodies are associated, the precise role of P-bodies in RNAi remains unclear. There is evidence that P-body components are essential for miRNA-based gene silencing. Depletion of certain P-body components, including RCK/P54 (Chu and Rana, 2006) and GW182 in human (Liu et al., 2005a) and Drosophila (Rehwinkel et al., 2005) cells leads to a loss of silencing. Furthermore, the decay of miRNA targets seems to require the de...

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“…In the other pathway, shortening of the poly(A) tail leads to the removal of the cap structure of the mRNA by the decapping proteins DCP1 and DCP2 facilitating 5Ј-3Ј degradation of the mRNA through the exoribonuclease XRN1 (Beelman and Parker, 1995;Butler, 2002). These proteins colocalize in discrete cytoplasmic foci in mammalian cells, termed processing bodies or P-bodies (also known as DCP1-or GW182-bodies), indicating that mRNA decay is restricted to distinct cytoplasmic compartments in mammalian cells (van Dijk et al, 2002;Cougot et al, 2004). The human protein CCR4, which is involved in mRNA deadenylation, the decapping stimulating LSm proteins LSm1-7, the DEAD/Hbox RNA helicase DDX6 (also known as RCK/p54), GW182, and Ge-1 are components of P-bodies (Bouveret et al, 2000;Eystathioy et al, 2002;Ingelfinger et al, 2002; LykkeAndersen, 2002;Cougot et al, 2004;Yu et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

“…Images were taken using Zeiss software LSM 5 version 3.5. The following antibodies were used: TIA-1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), DDX6 (1:500; Novus Biologicals), hemagglutinin (1:500; Roche Diagnostics), FLAG (1:500; Sigma Chemie), DCP1 (1:600; van Dijk et al, 2002), PABP (1:500; (Kuyumcu-Martinez et al, 2004), ATXN2 (1:200; BD Biosciences); MYC (1:600; Upstate Biotechnology, Lake Placid, NY); ␣-goat-Cy3 or ␣-rabbit-Cy3 (1:500; Dianova, Hamburg, Germany), and ␣-mouse-fluorescein isothiocyanate (FITC) (1:500; Dianova). …”

mentioning

confidence: 99%

Ataxin-2 Interacts with the DEAD/H-Box RNA Helicase DDX6 and Interferes with P-Bodies and Stress Granules

Nonhoff

Ralser

Welzel

et al. 2007

MBoC

314

320

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Tight control of translation is fundamental for eukaryotic cells, and deregulation of proteins implicated contributes to numerous human diseases. The neurodegenerative disorder spinocerebellar ataxia type 2 is caused by a trinucleotide expansion in the SCA2 gene encoding a lengthened polyglutamine stretch in the gene product ataxin-2, which seems to be implicated in cellular RNA-processing pathways and translational regulation. Here, we substantiate a function of ataxin-2 in such pathways by demonstrating that ataxin-2 interacts with the DEAD/H-box RNA helicase DDX6, a component of P-bodies and stress granules, representing cellular structures of mRNA triage. We discovered that altered ataxin-2 levels interfere with the assembly of stress granules and cellular P-body structures. Moreover, ataxin-2 regulates the intracellular concentration of its interaction partner, the poly(A)-binding protein, another stress granule component and a key factor for translational control. Thus, our data imply that the cellular ataxin-2 concentration is important for the assembly of stress granules and P-bodies, which are main compartments for regulating and controlling mRNA degradation, stability, and translation. INTRODUCTIONAn essential step in the control of global gene expression in eukaryotes is the regulation of mRNA translation and degradation. Shortening of the poly(A) tail is the initial step to trigger mRNA for decay in two major mRNA degradation pathways in eukaryotic cells (Bernstein and Ross, 1989;Peltz et al., 1991;Decker and Parker, 1994;Beelman and Parker, 1995). In one pathway, the deadenylated mRNA is degraded by a cytoplasmic protein complex, the exosome, consisting of a number of exonucleases with 3Ј-5Ј activity (Butler, 2002). In the other pathway, shortening of the poly(A) tail leads to the removal of the cap structure of the mRNA by the decapping proteins DCP1 and DCP2 facilitating 5Ј-3Ј degradation of the mRNA through the exoribonuclease XRN1 (Beelman and Parker, 1995;Butler, 2002). These proteins colocalize in discrete cytoplasmic foci in mammalian cells, termed processing bodies or P-bodies (also known as DCP1-or GW182-bodies), indicating that mRNA decay is restricted to distinct cytoplasmic compartments in mammalian cells (van Dijk et al., 2002;Cougot et al., 2004). The human protein CCR4, which is involved in mRNA deadenylation, the decapping stimulating LSm proteins LSm1-7, the DEAD/Hbox RNA helicase DDX6 (also known as RCK/p54), GW182, and Ge-1 are components of P-bodies (Bouveret et al., 2000;Eystathioy et al., 2002;Ingelfinger et al., 2002; LykkeAndersen, 2002;Cougot et al., 2004;Yu et al., 2005). Moreover, the RNA-associated protein 55, hEDC3, Hedls as well as factors of the RISC complex localize to P-bodies in mammalian cells (Fenger-Gron et al., 2005;Liu et al., 2005;Sen and Blau, 2005;Yang et al., 2006). P-bodies represent dynamic structures that are in an equilibrium with polysomes and contain nontranslating mRNA . Remarkably, recent work demonstrated that mRNA molecules entering P-bodies can a...

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Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures

Cited by 427 publications

References 37 publications

Small Interfering RNA-mediated Silencing Induces Target-dependent Assembly of GW/P Bodies

Small Interfering RNA-mediated Silencing Induces Target-dependent Assembly of GW/P Bodies

Localization of Double-stranded Small Interfering RNA to Cytoplasmic Processing Bodies Is Ago2 Dependent and Results in Up-Regulation of GW182 and Argonaute-2

Ataxin-2 Interacts with the DEAD/H-Box RNA Helicase DDX6 and Interferes with P-Bodies and Stress Granules

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