Getting to the Root of miRNA-Mediated Gene Silencing

Eulálio, Ana; Huntzinger, Eric; Izaurralde, Elisa

doi:10.1016/j.cell.2007.12.024

Cited by 955 publications

(716 citation statements)

References 33 publications

(85 reference statements)

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“…The up-regulation of GW182 is more intriguing, because GW182's exact function is yet to be determined. In Drosophila, Ago1-GW182 complexes are essential for miRNA-mediated silencing (Eulalio et al, 2008b), although how this complex achieves silencing is still debated (Eulalio et al, 2008a;Wu and Belasco, 2008). Tethering of GW182 to target mRNA shows that GW182 may facilitate silencing through a mode that bypasses the requirement for Ago2 (Behm-Ansmant et al, 2006).…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…They control a wide range of biological functions such as cellular proliferation, differentiation and apoptosis (Zhao and Srivastava, 2007;Erson and Petty, 2008;Eulalio et al, 2008). Recent reports showed strong evidence that miRNAs can act as oncogenes or tumor suppressors, having key roles in cancer initiation and progression (Cho, 2007;Tili et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

Regulation of Axl receptor tyrosine kinase expression by miR-34a and miR-199a/b in solid cancer

et al. 2011

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Axl is a receptor that induces proliferation, migration and invasion in cancer. In this study, we show that specific microRNAs (miRNAs) target the 3 0 -UTR of Axl. Luciferase-reporter assays with wild-type and deleted miR-34 and miR-199a/b seed sequences of Axl 3 0 -UTR confirmed the specificity of targeting. An inverse correlation between Axl protein and miR-34a expression in a panel of non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and breast cancer (BRC) cell lines was observed, while miR-199a/b expression was completely suppressed. Pre-miR transfection inhibited in vitro migration and invasion and, in vivo, reduced the number of distant lung-or liver-metastases in a chorion-allantoicmembrane (CAM) assay. Moreover, methylation-specific PCR on bisulfite-converted DNA obtained from the cell lines showed that the miR-34a promoter methylation status was inversely correlated with its expression, and that miR-199a/b promoter regions were methylated in all cells tested. In a panel of NSCLC tissues (n ¼ 44), miR34a and miR-199a/b were found to be downregulated and significantly co-expressed. A lower expression of all three miRs was significantly associated with squamous histotypes, and, in a preliminary series, NSCLC patients with miR-34a upregulation showed a positive association towards a longer survival. These results indicate that Axl receptor expression can be regulated by miR-34a and miR-199a/b, which are suppressed by promoter methylation in solid cancer cells.

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“…Mature miRNAs are produced from sequential processing of primary transcripts via specific RNAses. These 18-to 24-nt-long miRNAs down-regulate protein expression of specific mRNA by either translational inhibition or mRNA degradation [14]. Numerous reports have now shown that miRNAs are differentially expressed in many types of human cancers [15,16].…”

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confidence: 99%

“…In the RISC complex, the active strand is retained and the passenger strand is selectively degraded. The mature miRNA along with RISC binds to complementary sites in the mRNA transcripts and (typically) downregulates gene expression [14,19]. miRNAs that bind to mRNA targets with imperfect complementarity regulate the target gene at the level of protein translation, while miRNAs that bind to their mRNA targets with perfect complementarity induce degradation [20,21].…”

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confidence: 99%