Multiple independent domains of dGW182 function in miRNA-mediated repression in <i>Drosophila</i>

Chekulaeva, Marina; Filipowicz, Witold; Parker, Roy

doi:10.1261/rna.1364909

Cited by 67 publications

(119 citation statements)

References 57 publications

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“…4c, black bars), and depletion of dGW182 (open bars) partially alleviated miR-9b-induced repression; as expected, transfection of a plasmid encoding wild-type dGW182 resistant to RNAi rescued the repression. Mutations of tryptophans in either NED (6W) or CED (7W) had only a minor effect on the functionality of dGW182 in the rescue, consistent with independent repression by NED and CED domains 5 . However, combining the tryptophan mutations in both regions led to a strong alleviation of repression, demonstrating the role of W-motifs in miRNA-mediated silencing.…”

Section: W-motifs Function In a Genuine Mirna-mediated Repressionsupporting

confidence: 55%

“…2c). The ability of both mutants to still partially repress mRNA function and associate with CCR4-NOT is readily explained by observations that, in addition to the CED, N-proximal regions of GW182s have the potential to repress mRNAs 5,9,17,18 and associate with CCR4-NOT components (see below).…”

Section: Repression By the Ced Correlates With Ccr 4-not Interactionmentioning

confidence: 99%

“…In mammals, tethering of the three regions mentioned above also represses reporter mRNA, with the major contribution being provided by the CED [9][10][11] . The mechanism by which GW182 domains repress mRNA function appears to be evolutionarily conserved, as dGW182 can repress mRNA function in mammalian cells, and human TNRC6 proteins (mammals express three counterparts of dGW182: TNRC6A, B and C) act as repressors in D. melanogaster cells 5,8,9 .…”

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confidence: 99%

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miRNA repression involves GW182-mediated recruitment of CCR4–NOT through conserved W-containing motifs

Chekulaeva

Mathys

Zipprich

et al. 2011

Nat Struct Mol Biol

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AbstractmiRNA-mediated repression in animals is dependent on the GW182 protein family. GW182 proteins are recruited to the miRNA repression complex through direct interaction with Argonaute proteins, and they function downstream to repress target mRNA. Here we demonstrate that in human and Drosophila melanogaster cells, the critical repressive features of both the N-terminal and C-terminal effector domains of GW182 proteins are Gly/Ser/Thr-Trp (G/S/TW) or Trp-Gly/ Ser/Thr (WG/S/T) motifs. These motifs, which are dispersed across both domains and act in an additive manner, function by recruiting components of the CCR 4-NOT deadenylation complex. A heterologous yeast polypeptide with engineered WG/S/T motifs acquired the ability to repress tethered mRNA and to interact with the CCR 4-NOT complex. These results identify previously unknown effector motifs functioning as important mediators of miRNA-induced silencing in both species, and they reveal that recruitment of the CCR 4-NOT complex by tryptophan-containing motifs acts downstream of GW182 to repress mRNA s, including inhibiting translation independently of deadenylation.MicroRNAs (miRNAs) are small, ~21-nt-long RNAs that post-transcriptionally regulate gene expression in eukaryotes. In animals, miRNAs bind to partially complementary sites in mRNAs, leading to translational repression and mRNA deadenylation and degradation [1][2][3][4] . miRNAs function as part of ribonucleoprotein complexes, miRNPs, with Argonaute (AGO) and GW182 family proteins being the crucial components. GW182s interact directly with AGO proteins and function downstream as effectors mediating mRNA repression. Hence, understanding the function of GW182 proteins is critical for understanding miRNAmediated repression.GW182 functional regions have been mapped in D. melanogaster and mammalian proteins. In D. melanogaster, three regions were found to repress tethered mRNA to a similar extent 5

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Section: W-motifs Function In a Genuine Mirna-mediated Repressionsupporting

confidence: 55%

Section: Repression By the Ced Correlates With Ccr 4-not Interactionmentioning

confidence: 99%

mentioning

confidence: 99%

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miRNA repression involves GW182-mediated recruitment of CCR4–NOT through conserved W-containing motifs

Chekulaeva

Mathys

Zipprich

et al. 2011

Nat Struct Mol Biol

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324

449

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“…In their N-terminal half, GW proteins contain multiple GW repeats, which directly interact with an Ago protein. This domain is commonly referred to as the Ago-binding domain, and GW repeats have been suggested to function as so called "Ago-hooks" (13)(14)(15)(16)(17). The C-terminal half of mammalian GW proteins contains two globular domains and several sequence motifs, which are embedded into presumably unstructured spacer regions.…”

mentioning

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%

Structural features of Argonaute–GW182 protein interactions

Pfaff

Hennig

Herzog

et al. 2013

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

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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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microRNA strand selection: Unwinding the rules

2020

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microRNAs (miRNAs) play a central role in the regulation of gene expression by targeting specific mRNAs for degradation or translational repression. Each miRNA is post‐transcriptionally processed into a duplex comprising two strands. One of the two miRNA strands is selectively loaded into an Argonaute protein to form the miRNA‐Induced Silencing Complex (miRISC) in a process referred to as miRNA strand selection. The other strand is ejected from the complex and is subject to degradation. The target gene specificity of miRISC is determined by sequence complementarity between the Argonaute‐loaded miRNA strand and target mRNA. Each strand of the miRNA duplex has the capacity to be loaded into miRISC and possesses a unique seed sequence. Therefore, miRNA strand selection plays a defining role in dictating the specificity of miRISC toward its targets and provides a mechanism to alter gene expression in a switch‐like fashion. Aberrant strand selection can lead to altered gene regulation by miRISC and is observed in several human diseases including cancer. Previous and emerging data shape the rules governing miRNA strand selection and shed light on how these rules can be circumvented in various physiological and pathological contexts. This article is categorized under: RNA Processing > Processing of Small RNAs Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs

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Multiple independent domains of dGW182 function in miRNA-mediated repression in Drosophila

Cited by 67 publications

References 57 publications

miRNA repression involves GW182-mediated recruitment of CCR4–NOT through conserved W-containing motifs

miRNA repression involves GW182-mediated recruitment of CCR4–NOT through conserved W-containing motifs

Structural features of Argonaute–GW182 protein interactions

microRNA strand selection: Unwinding the rules

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