Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity

Rand, Tim A.; Ginalski, Krzysztof; Grishin, Nick V.; Wang, Xiaodong

doi:10.1073/pnas.0405913101

Cited by 282 publications

(203 citation statements)

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“…9 Ago2 may function as an RNA-induced silencing complex (RISC) slicer by way of its RNase activity, cleaving targeted messenger RNA (mRNA)s that are complementary to the guiding small interference RNA (siRNA) or microRNA (miRNA). [10][11][12][13] Ago2 can specifically generate an additional miRNA precursor, ac-pre-miRNA, which harbors a single strand cleavage in the 3 0 -arm of the premiRNA, and thus form a shortened hairpin bound to a fragment of about 11-12 nucleotides. 14 Most interestingly, the Ago family has recently been found to be in human tumors.…”

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confidence: 99%

Argonaute2 promotes tumor metastasis by way of up-regulating focal adhesion kinase expression in hepatocellular carcinoma

Cheng

Han

2013

Hepatology

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Hepatocellular carcinoma (HCC) is one of the most common cancers and shows a propensity to metastasize and infiltrate adjacent and more distant tissues. However, the mechanisms that contribute to tumor metastasis remain unclear. Here we evaluate the effect of Argonaute2 (Ago2), a member of the Ago gene family that plays a role in short interfering RNA-mediated gene silencing, on HCC tumorigenesis, and metastasis. We found that Ago2 was frequently up-regulated in HCC specimens compared to that in corresponding adjacent nontumor liver. Interestingly, Ago2 overexpression can promote proliferation, colony formation in an anchor-independent manner, migration, tumorigenicity, and metastasis of HCC cells in vivo; in contrast, Ago2 knockdown can restrict anchor-independent colony formation, migration, and tumor metastasis of HCC cells in vivo. However, known microRNAs related to tumor metastasis appeared not be deregulated with Ago2 overexpression in HCC cells; even the knockdown of Dicer, which is responsible for micro-RNA biosynthesis, did not abolish the actions of Ago2 in HCC cells. Significantly, focal adhesion kinase (FAK), a well-known molecule associated with tumor metastasis, was upregulated as a result of Ago2 overexpression. Chromatin immunoprecipitation assay showed that Ago2 can bind to the FAK promoter and then trigger its transcription. Moreover, an increased DNA copy number of Ago2 on chromosome 8q24, one of the most frequent DNA amplified regions, was validated and shown by way of fluorescence in situ hybridization. Conclusion: Our data demonstrate that Ago2 overexpression, as a result of genomic DNA amplification, promotes HCC tumorigenesis and metastasis by way of upregulation of FAK transcription, thereby providing new insight into HCC progression and

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confidence: 99%

Argonaute2 promotes tumor metastasis by way of up-regulating focal adhesion kinase expression in hepatocellular carcinoma

Cheng

Han

2013

Hepatology

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“…Argonautes, which contain conserved PAZ and PIWI domains, seem to be the sole proteins required for RISC-mediated activities 24 . Active RISCs, which contain only a single guide RNA strand 25 , are capable of directing multiple rounds of site-specific target mRNA cleavage 26 .…”

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confidence: 99%

Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

Yuan

Meister

et al. 2005

Nature

571

526

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RNA interference (RNAi) is a conserved sequence-specific gene regulatory mechanism 1-3 mediated by the RNA-induced silencing complex (RISC), which is composed of a single-stranded guide RNA and an Argonaute protein. The PIWI domain, a highly conserved motif within Argonaute, has been shown to adopt an RNase H fold 4,5 critical for the endonuclease cleavage activity of RISC 4-6 . Here we report the crystal structure of Archaeoglobus fulgidus Piwi protein bound to double-stranded RNA, thereby identifying the binding pocket for guide-strand 5′-end recognition and providing insight into guide-strand-mediated messenger RNA target recognition. The phosphorylated 5′ end of the guide RNA is anchored within a highly conserved basic pocket, supplemented by the carboxy-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5′ end of the guide RNA is unpaired and stacks over a conserved tyrosine residue, whereas successive nucleotides form a four-base-pair RNA duplex. Mutation of the corresponding amino acids that contact the 5′ phosphate in human Ago2 resulted in attenuated mRNA cleavage activity. Our structure of the Piwi-RNA complex, and that determined elsewhere 7 , provide direct support for the 5′ region of the guide RNA serving as a nucleation site for pairing with target mRNA and for a fixed distance separating the RISC-mediated mRNA cleavage site from the anchored 5′ end of the guide RNA.Small interfering RNAs (siRNAs), the cleavage products of RNase III enzyme Dicer, consist of RNA duplexes that contain two-nucleotide 3′ overhangs and 5′-phosphorylated ends. These unique features at the termini of 19-23-base-pair duplexes, which distinguish siRNAs from other endogenous RNAs, are also required for their functional role in RNAi-mediated processes. Extensive structural and biochemical studies on PAZ (for PIWI/Argonaute/ Zwille)-RNA recognition [8][9][10] , culminating in the structures of PAZ bound to singlestranded RNA 11 and siRNA-like duplexes 12 , have established that the 3′ overhang isCorrespondence and requests for materials should be addressed to D.J.P. (pateld@mskcc.org).. Supplementary Information accompanies the paper on www.nature.com/nature. Competing interests statementThe authors declare that they have no competing financial interests.Coordinates have been deposited in the Protein Data Bank under accession code 1YTU. Here we describe the 2.5-Å crystal structure of the A. fulgidus Piwi protein (Fig. 1a) bound to a 5′-phosphate-containing 21-mer RNA, capable of self-complementary duplex formation (Fig. 1b). The crystallographic asymmetric unit contains two Piwi proteins, each bound to a short four-base-pair segment of duplex, positioned adjacent to the 5′ phosphate and its attached unpaired nucleotide (Fig. 1c). The structure of the A. fulgidus Piwi protein, which consists of a short N-terminal element and two domains labelled A and B (Fig. 1a), is similar (root-mean-square deviation = 0.65Å for Cα) in the free state 5 and in the RNAbound complex. The RNA, whose sequence ...

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“…The siRNA duplexes are loaded onto the RNAi effector, RNA-induced silencing complex (RISC). An Argonaute (Ago)-family protein forms the catalytic core of RISC (Hammond et al 2001;Liu et al 2004;Rand et al 2004;Song et al 2004). Before the activation of RISC, the siRNA duplex is dissociated and the passenger strand removed (Nykanen et al 2001;Okamura et al 2004;Tomari et al 2004).…”

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confidence: 99%

“…Before the activation of RISC, the siRNA duplex is dissociated and the passenger strand removed (Nykanen et al 2001;Okamura et al 2004;Tomari et al 2004). The guide strand of the siRNA remains in the RISC and directs cleavage of target mRNA (Hammond et al 2001;Liu et al 2004;Rand et al 2004;Song et al 2004;Rivas et al 2005).…”

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confidence: 99%

QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands

Maiti¹,

Lee²,

Liu³

2007

Genes Dev.

113

149

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Single-stranded small interfering RNA (siRNA) guides the cleavage of homologous mRNA by Argonaute proteins, the catalytic core of the RNA-induced silencing complex (RISC), in the conserved RNA interference (RNAi) pathway. The separation of the siRNA duplex into single strands is essential for the activation of RISC. Previous biochemical studies have suggested that Argonaute proteins cleave and remove the passenger strand of siRNA duplex from RISC, but the in vivo importance of this process and the mechanism for the removal of the nicked passenger strand are not known. Here, we show that in the filamentous fungus Neurospora, the Argonaute homolog QDE-2 and its slicer function are required for the generation of single-stranded siRNA and gene silencing in vivo. Biochemical purification of QDE-2 led to the identification of QIP, a QDE-2-interacting protein, with an exonuclease domain. The disruption of qip in Neurospora impaired gene silencing and siRNA accumulated, mostly in nicked duplex form. Furthermore, our results suggest that QIP acts as an exonuclease that cleaves and removes the nicked passenger strand from siRNA duplex in a QDE-2-dependent manner. Together, these results suggest that both the cleavage and removal of the passenger strand from the siRNA duplex are important steps in RNAi pathways.[Keywords: RNAi; Neurospora; Argonaute; siRNA; exonuclease; RISC] Supplemental material is available at http://www.genesdev.org.

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Biochemical identification of Argonaute 2 as the sole protein required for RNA-induced silencing complex activity

Cited by 282 publications

References 24 publications

Argonaute2 promotes tumor metastasis by way of up-regulating focal adhesion kinase expression in hepatocellular carcinoma

Argonaute2 promotes tumor metastasis by way of up-regulating focal adhesion kinase expression in hepatocellular carcinoma

Structural basis for 5′-end-specific recognition of guide RNA by the A. fulgidus Piwi protein

QIP, a putative exonuclease, interacts with the Neurospora Argonaute protein and facilitates conversion of duplex siRNA into single strands

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