A C-terminal silencing domain in GW182 is essential for miRNA function

Eulálio, Ana; Helms, Sigrun; Fritzsch, Christoph; Fauser, Maria; Izaurralde, Elisa

doi:10.1261/rna.1605509

Cited by 109 publications

(206 citation statements)

References 34 publications

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“…Using protein-protein cross-links, followed by mass spectrometry (MS), we map the GW-binding region on Ago2. Interestingly, we find that only two Trp residues are required for Ago2 binding, and all crosslinks surround two specific Trp-binding pockets (20). Our data allow us to put the requirement of tryptophans into the greater context of the Ago-binding domain and to propose a two-step binding mode for their interaction.…”

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confidence: 68%

“…Using truncation mutants, the binding domain was narrowed down to shorter sequence stretches. In Drosophila GW182, motif I and motif II were identified as the two main Ago-interaction platforms (16,20). Here, we used a peptide scanning approach to identify Ago-binding motifs in the mammalian GW homolog TNRC6B.…”

Section: Only Two Tryptophan Residues In Tnrc6b-599-683 Are Necessarymentioning

confidence: 99%

“…The C-terminal half of mammalian GW proteins contains two globular domains and several sequence motifs, which are embedded into presumably unstructured spacer regions. Because the C-terminal part is capable of silencing independently of Ago binding when artificially tethered to mRNAs, it is referred to as "silencing domain" (14,(17)(18)(19)(20). The Ago-binding domain and the silencing domain are separated by a region containing an ubiquitin-associated domain and a Qrich element, both of which with unknown functions.…”

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confidence: 99%

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Structural features of Argonaute–GW182 protein interactions

Pfaff

Hennig

Herzog

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

100

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MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to target mRNAs, leading to gene silencing. However, Ago proteins are not the actual mediators of gene silencing but interact with a member of the GW182 protein family (also known as GW proteins), which coordinates all downstream steps in gene silencing. GW proteins contain an N-terminal Ago-binding domain that is characterized by multiple GW repeats and a C-terminal silencing domain with several globular domains. Within the Ago-binding domain, Trp residues mediate the direct interaction with the Ago protein. Here, we have characterized the interaction of Ago proteins with GW proteins in molecular detail. Using biochemical and NMR experiments, we show that only a subset of Trp residues engage in Ago interactions. The Trp residues are located in intrinsically disordered regions, where flanking residues mediate additional weak interactions, that might explain the importance of specific tryptophans. Using cross-linking followed by mass spectrometry, we map the GW protein interactions with Ago2, which allows for structural modeling of Ago-GW182 interaction. Our data further indicate that the Ago-GW protein interaction might be a two-step process involving the sequential binding of two tryptophans separated by a spacer with a minimal length of 10 aa.

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confidence: 68%

Section: Only Two Tryptophan Residues In Tnrc6b-599-683 Are Necessarymentioning

confidence: 99%

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confidence: 99%

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Structural features of Argonaute–GW182 protein interactions

Pfaff

Hennig

Herzog

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

100

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“…We generated three classes of mutations: (i) mutations on the putative RNA-binding interface, (ii) mutations in the MID-PIWI interface, and (iii) deletions of either the eukaryote-specific C-terminal insertion or of loop L1 (Table S2). A Dm AGO1 F2V2 mutant that is unable to bind miRNAs and GW182 (27,29) served as a negative control.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

Boland

Huntzinger

Schmidt

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Argonaute proteins (AGOs) are essential effectors in RNAmediated gene silencing pathways. They are characterized by a bilobal architecture, in which one lobe contains the N-terminal and PAZ domains and the other contains the MID and PIWI domains. Here, we present the first crystal structure of the MID-PIWI lobe from a eukaryotic AGO, the Neurospora crassa QDE-2 protein.Compared to prokaryotic AGOs, the domain orientation is conserved, indicating a conserved mode of nucleic acid binding. The PIWI domain shows an adaptable surface loop next to a eukaryote-specific α-helical insertion, which are both likely to contact the PAZ domain in a conformation-dependent manner to sense the functional state of the protein. The MID-PIWI interface is hydrophilic and buries residues that were previously thought to participate directly in the allosteric regulation of guide RNA binding. The interface includes the binding pocket for the guide RNA 5′ end, and residues from both domains contribute to binding. Accordingly, micro-RNA (miRNA) binding is particularly sensitive to alteration in the MID-PIWI interface in Drosophila melanogaster AGO1 in vivo. The structure of the QDE-2 MID-PIWI lobe provides molecular and mechanistic insight into eukaryotic AGOs and has significant implications for understanding the role of these proteins in silencing.P roteins of the Argonaute (AGO) family play essential roles in RNA-mediated gene silencing mechanisms in eukaryotes (1, 2). They are loaded with small noncoding RNAs to form the core of RNA-induced silencing complexes, which repress the expression of target genes at the transcriptional or posttranscriptional level (1, 2). The targets to be silenced are selected through base-pairing interactions between the loaded small RNA (also known as the guide RNA) and an mRNA target containing partially or fully complementary sequences (1-3).Thus far, structural information on full-length AGOs has been available only for the homologous proteins from Archaea and Eubacteria, which preferentially use DNA as a guide (4-10). These studies revealed that AGOs consist of four domains: the N-terminal domain; the PAZ domain, which binds the 3′ end of guide RNAs/DNAs; the MID domain, which provides a binding pocket for the 5′ phosphate of guide RNAs/DNAs; and the PIWI domain, which adopts an RNase H fold and has endonucleolytic activity in some, but not all, AGOs (4-11).For the eukaryotic AGO clade of Argonaute proteins, structural information is available only for the isolated PAZ domains of Drosophila melanogaster (Dm) AGO1 and AGO2, human AGO1 (12-16) and the MID domains of human AGO2 and Neurospora crassa (Nc) QDE-2 (17, 18). Structural information is also available for PAZ domains of the PIWI clade of AGOs (19,20). These studies showed that the PAZ and MID domains of eukaryotic AGOs adopt folds similar to the prokaryotic homologs and recognize the 3′-and 5′-terminal nucleotides of the guide strand, respectively, in a similar manner to their prokaryotic counterparts (12-18).Our previous structure of the isolate...

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“…10 Taken together, these results indicate that miRNA-mediated gene silencing is achieved by AGO1 in complex with GW182 independent of its localization to P-bodies. 12 Megha Ghildiyal from Phil Zamore's group (University of Massachusetts, Worcester, MA) presented her recently published findings that in Drosophila melanogaster small RNAs are distributed among Ago1 and Ago2 according to their duplex structure and the identity of their first nucleotide. Thus, perfectly matched double-stranded RNAs, like siRNAs, associate with Ago2, whereas central mismatches, as can be found in miRNA/ miRNA* duplexes, lead to the association with Ago1.…”

Section: The Argonaute Worldmentioning

confidence: 99%