P-Body Formation Is a Consequence, Not the Cause, of RNA-Mediated Gene Silencing

Eulálio, Ana; Behm‐Ansmant, Isabelle; Schweizer, Daniel; Izaurralde, Elisa

doi:10.1128/mcb.00128-07

Cited by 619 publications

(765 citation statements)

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“…Nonetheless, their data also implicated differences in the regulation of GWB formation in Drosophila from that in human. For example, Ago2-knockdown disassembled GWB and long double-stranded RNA did not restore GWB in LSm1-knockdown or rck/p54-knockdown cells in Drosophila (Eulalio et al, 2007b). These apparent discrepancies with our data may be attributed to the potential difference in the function of GWB components and in the RNAi pathway between Drosophila and human cells (Lee et al, 2004;Okamura et al, 2004).…”

Section: Regulation Of Gwb Assemblycontrasting

confidence: 95%

“…We speculate that under certain circumstances, GWB may serve as markers for siRNA/miRNA activity; therefore, the variation in number and size of GWB may correlate with the activities of miRNA during different stages of the cell cycle and proliferation (Yang et al, 2004;Hatfield et al, 2005;He et al, 2005;O'Donnell et al, 2005;Lian et al, 2006). Interestingly, a recent publication reported that Drosophila siRNA:mRNA or miRNA:mRNA also nucleated the formation of GWB (Eulalio et al, 2007b), an observation that strongly supports our finding that siRNA/miRNA-mediated function is a key regulatory mechanism of GWB assembly. Nonetheless, their data also implicated differences in the regulation of GWB formation in Drosophila from that in human.…”

Section: Regulation Of Gwb Assemblymentioning

confidence: 93%

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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: Regulation Of Gwb Assemblycontrasting

confidence: 95%

Section: Regulation Of Gwb Assemblymentioning

confidence: 93%

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

Lian¹,

Fritzler

Katz³

et al. 2007

MBoC

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“…However, recent studies indicate that mRNA decay, NMD and RNAi-mediated gene silencing are functional in Drosophila and mammalian cells lacking detectable microscopic P bodies, suggesting that the formation of P bodies is a consequence of these processes (Chu and Rana, 2006;Eulalio et al, 2007). Here we revealed that a large number of genes whose inactivations enhance the formation of P bodies are also involved in regulating NMD, RNAi-and miRNA-mediated gene silencing, providing a link between the formation of P bodies and these RNA metabolism processes.…”

Section: Role Of Translation In Regulating Rnaimentioning

confidence: 99%

A genome-wide RNAi screen identifies genes regulating the formation of P bodies in C. elegans and their functions in NMD and RNAi

et al. 2011

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Cytoplasmic processing bodies, termed P bodies, are involved in diverse post-transcriptional processes including mRNA decay, nonsense-mediated RNA decay (NMD), RNAi, miRNA-mediated translational repression and storage of translationally silenced mRNAs. Regulation of the formation of P bodies in the context of multicellular organisms is poorly understood. Here we describe a systematic RNAi screen in C. elegans that identified 224 genes with diverse cellular functions whose inactivations result in a dramatic increase in the number of P bodies. 83 of these genes form a complex functional interaction network regulating NMD. We demonstrate that NMD interfaces with many cellular processes including translation, ubiquitin-mediated protein degradation, intracellular trafficking and cytoskeleton structure. We also uncover an extensive link between translation and RNAi, with different steps in protein synthesis appearing to have distinct effects on RNAi efficiency. Moreover, the intracellular vesicular trafficking network plays an important role in the regulation of RNAi. A subset of genes enhancing P body formation also regulate the formation of stress granules in C. elegans. Our study offers insights into the cellular mechanisms that regulate the formation of P bodies and also provides a framework for system-level understanding of NMD and RNAi in the context of the development of multicellular organisms.

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“…Indeed, depletion of P-body components such as LSm1 and RCK/p54 (Chu and Rana, 2006) in human cells has no effect on siRNA silencing, leading to a view that P-bodies are dispensable for siRNA-mediated RNAi. However, depletion of the P-body structural component GW182 has shown mixed results; three reports show that silencing GW182 does inhibit siRNA silencing to a certain extent (Jakymiw et al, 2005;Liu et al, 2005a;Lian et al, 2007), whereas others show no requirement of GW182 for siRNA function (Rehwinkel et al, 2005;Chu and Rana, 2006;Eulalio et al, 2007b). However, siRNAs have been found to localize to P-bodies (Jakymiw et al, 2005), and the number and size of P-bodies were found to increase upon siRNA transfection, in a target-dependant manner (Lian et al, 2007 Lsm1 or RCK/p54, functional siRNAs are capable of inducing P-body reassembly (Lian et al, 2007).…”

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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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P-Body Formation Is a Consequence, Not the Cause, of RNA-Mediated Gene Silencing

Cited by 619 publications

References 64 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

A genome-wide RNAi screen identifies genes regulating the formation of P bodies in C. elegans and their functions in NMD and RNAi

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

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