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

Lian, Shangli; Fritzler, Marvin J.; Katz, Joseph; Hamazaki, Takashi; Terada, Naohiro; Satoh, Minoru; Chan, Edward K. L.

doi:10.1091/mbc.e07-01-0070

Cited by 42 publications

(55 citation statements)

References 50 publications

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“…We confirmed the level of Ago2 protein knockdown at 90% by measuring knockdown on an eGFP reporter (data not shown). It has been reported previously that Ago2 knockdown has no noticeable effect on P-body integrity or number in mammalian cells, whereas knockdown of GW182 and RCK/p54 leads to the disappearance of P-bodies, and our observations confirmed the same ( Figure 3B) (Lian et al, 2007). We then transfected Cy3-siPPIB into these cells and observed the localization pattern of siRNAs.…”

Section: Ago2 Is Required For Localization And/or Retention Of Sirnassupporting

confidence: 90%

“…We knocked down Ago2, GW182, and RCK/p54 in HeLa cells by using siRNA transfection (Chu and Rana, 2006;Lian et al, 2007), and we found high levels of knockdown in all cases (Ͼ80%) ( Figure 3A). We confirmed the level of Ago2 protein knockdown at 90% by measuring knockdown on an eGFP reporter (data not shown).…”

Section: Ago2 Is Required For Localization And/or Retention Of Sirnasmentioning

confidence: 96%

“…All other siRNAs were obtained from Eurogentec (Seraing, Belgium). All sequences 5Ј-3Ј, sense strand only, are as follows: siPPIB, GGA-AAG-ACU-GUU-CCA-AAA-AUU (Dharmacon RNA Technologies); siLA, GGU-GGU-GAC-GAU-CUG-GGC-UUU (Dharmacon RNA Technologies); siAgo2, GCA-CGG-AAG-UCC-AUC-UGA-AUU (Chu and Rana, 2006); siLuc, UAA-GGC-UAU-GAA-GAG-AUA-CUU (Dharmacon RNA Technologies); siGW, GAA AUG CUC UGG UCC GCU AUU (Lian et al, 2007); siRCK, GCA GAA ACC CUA UGA GAU UUU (Chu and Rana, 2006); siSMN, GCA UGC UCU AAA GAA UGG UUU; and si-MAPK (AllStars Positive control Mm/Hs MAPK1 control siRNA; QIAGEN). DNA Constructs.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

“…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).…”

Section: Introductionmentioning

confidence: 99%

“…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). Moreover, after knockdown of Lsm1 or RCK/p54, functional siRNAs are capable of inducing P-body reassembly (Lian et al, 2007). This suggests that although microscopic P-bodies are not necessarily required for RNAi, silencing could occur in submicroscopic complexes that might then trigger the assembly of larger, microscopic structures.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Ago2 Is Required For Localization And/or Retention Of Sirnassupporting

confidence: 90%

Section: Ago2 Is Required For Localization And/or Retention Of Sirnasmentioning

confidence: 96%

Section: Cell Culture and Transfectionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

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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Silencing of ADAM33 restrains proliferation and induces apoptosis of airway smooth muscle cells in ovalbumin‐induced asthma model

Zhou

Bai

Liu

et al. 2018

J of Cellular Biochemistry

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A defibrinogen and metalloproteinase 33 (ADAM33) was reported to play an important role in asthma. Furthermore, ADAM33 may play a possible role in airway remodeling due to its high expression in myo‐/fibroblasts, epithelium, as well as the airway smooth muscle cells (ASMCs). Thus, the study is supposed to investigate the effect of the downregulation of ADAM33 on the proliferation and apoptosis of ASMCs in allergic asthma. An ovalbumin‐induced asthma model in rats was established for investigating the function of the silencing of ADAM33. ASMCs were cultured and divided into four groups after transfection. The messenger RNA and protein expressions of ADAM33 were measured by reverse transcription quantitative polymerase chain reaction and Western blot analysis. Cell proliferation was tested by cell counting kit‐8 and cell apoptosis by TdT‐mediated dUTP nick‐end labeling. The allergic asthma rats showed a large number of inflammatory cell infiltration, airway smooth muscle hypertrophy and hyperplasia, and increased WA t, WA m, and numbers of bronchial smooth muscle nucleus. Additionally, increased numbers of eosinophils and neutrophils, expressions of immunoglobulin E and interleukin‐4, content of airway air pressure, and NO, although decreased in expression of interferon‐γ, were exhibited in rats with allergic asthma. In our study, upregulated ADAM33 was found, and after the silencing of ADAM33, decreased proliferation and increased apoptosis of ASMCs were observed. The study evidences that silencing of ADAM33 can decrease the proliferation and increase the apoptosis of ASMCs in a rat model of allergic asthma, suggesting ADAM33 represents a potential investigative focus target aiding allergic asthma.

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Reflections on Ten Years of History of, and Future Prospects for, GW182 and GW/P Body Research

Chan

Yao

Fritzler

2012

Advances in Experimental Medicine and Biology

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

Cited by 42 publications

References 50 publications

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

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

Silencing of ADAM33 restrains proliferation and induces apoptosis of airway smooth muscle cells in ovalbumin‐induced asthma model

Reflections on Ten Years of History of, and Future Prospects for, GW182 and GW/P Body Research

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