Joerg E. Braun

GW182 proteins directly recruit cytoplasmic deadenylase complexes to miRNA targets.

miRNAs are posttranscriptional regulators of gene expression that associate with Argonaute and GW182 proteins to repress translation and/or promote mRNA degradation. miRNA-mediated mRNA degradation is initiated by deadenylation, although it is not known whether deadenylases are recruited to the mRNA target directly or by default, as a consequence of a translational block. To answer this question, we performed a screen for potential interactions between the Argonaute and GW182 proteins and subunits of the two cytoplasmic deadenylase complexes. We found that human GW182 proteins recruit the PAN2-PAN3 and CCR4-CAF1-NOT deadenylase complexes through direct interactions with PAN3 and NOT1, respectively. These interactions are critical for silencing and are conserved in D. melanogaster. Our findings reveal that GW182 proteins provide a docking platform through which deadenylase complexes gain access to the poly(A) tail of miRNA targets to promote their deadenylation, and they further indicate that deadenylation is a direct effect of miRNA regulation.

Structural Basis for the Mutually Exclusive Anchoring of P Body Components Edc3 and Tral to the Dead Box Protein Ddx6/Me31B.

The DEAD box helicase DDX6/Me31B functions in translational repression and mRNA decapping. How particular RNA helicases are recruited specifically to distinct functional complexes is poorly understood. We present the crystal structure of the DDX6 C-terminal RecA-like domain bound to a highly conserved FDF sequence motif in the decapping activator EDC3. The FDF peptide adopts an alpha-helical conformation upon binding to DDX6, occupying a shallow groove opposite to the DDX6 surface involved in RNA binding and ATP hydrolysis. Mutagenesis of Me31B shows the relevance of the FDF interaction surface both for Me31Bs accumulation in P bodies and for its ability to repress the expression of bound mRNAs. The translational repressor Tral contains a similar FDF motif. Together with mutational and competition studies, the structure reveals why the interactions of Me31B with EDC3 and Tral are mutually exclusive and how the respective decapping and translational repressor complexes might hook onto an mRNA substrate.

Two PABPC1‐binding sites in GW182 proteins promote miRNA‐mediated gene silencing

miRNA‐mediated gene silencing requires the GW182 proteins, which are characterized by an N‐terminal domain that interacts with Argonaute proteins (AGOs), and a C‐terminal silencing domain (SD). In Drosophila melanogaster (Dm) GW182 and a human (Hs) orthologue, TNRC6C, the SD was previously shown to interact with the cytoplasmic poly(A)‐binding protein (PABPC1). Here, we show that two regions of GW182 proteins interact with PABPC1: the first contains a PABP‐interacting motif 2 (PAM2; as shown before for TNRC6C) and the second contains the M2 and C‐terminal sequences in the SD. The latter mediates indirect binding to the PABPC1 N‐terminal domain. In D. melanogaster cells, the second binding site dominates; however, in HsTNRC6A–C the PAM2 motif is essential for binding to both Hs and DmPABPC1. Accordingly, a single amino acid substitution in the TNRC6A–C PAM2 motif abolishes the interaction with PABPC1. This mutation also impairs TNRC6s silencing activity. Our findings reveal that despite species‐specific differences in the relative strength of the PABPC1‐binding sites, the interaction between GW182 proteins and PABPC1 is critical for miRNA‐mediated silencing in animal cells.

The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets

Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, we characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases.

The role of GW182 proteins in miRNA-mediated gene silencing.

GW182 family proteins are essential for microRNA-mediated gene silencing in animal cells. They are recruited to miRNA targets through direct interactions with Argonaute proteins and promote target silencing. They do so by repressing translation and enhancing mRNA turnover. Although the precise mechanism of action of GW182 proteins is not fully understood, these proteins have been shown to interact with the cytoplasmic poly(A)-binding protein (PABP) and with the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These findings suggest that GW182 proteins function as scaffold proteins for the assembly of the multiprotein complex that silences miRNA targets.

A direct interaction between DCP1 and XRN1 couples mRNA decapping to 5′ exonucleolytic degradation

The removal of the mRNA 5′ cap structure by the decapping enzyme DCP2 leads to rapid 5′→3′ mRNA degradation by XRN1, suggesting that the two processes are coordinated, but the coupling mechanism is unknown. DCP2 associates with the decapping activators EDC4 and DCP1. Here we show that XRN1 directly interacts with EDC4 and DCP1 in human and Drosophila melanogaster cells, respectively. In D. melanogaster cells, this interaction is mediated by the DCP1 EVH1 domain and a DCP1-binding motif (DBM) in the XRN1 C-terminal region. The NMR structure of the DCP1 EVH1 domain bound to the DBM reveals that the peptide docks at a conserved aromatic cleft, which is used by EVH1 domains to recognize proline-rich ligands. Our findings reveal a role for XRN1 in decapping and provide a molecular basis for the coupling of decapping to 5′→3′ mRNA degradation.

The Structural Basis of Edc3- and Scd6-Mediated Activation of the Dcp1:Dcp2 Mrna Decapping Complex.

The Dcp1:Dcp2 decapping complex catalyses the removal of the mRNA 5′ cap structure. Activator proteins, including Edc3 (enhancer of decapping 3), modulate its activity. Here, we solved the structure of the yeast Edc3 LSm domain in complex with a short helical leucine‐rich motif (HLM) from Dcp2. The motif interacts with the monomeric Edc3 LSm domain in an unprecedented manner and recognizes a noncanonical binding surface. Based on the structure, we identified additional HLMs in the disordered C‐terminal extension of Dcp2 that can interact with Edc3. Moreover, the LSm domain of the Edc3‐related protein Scd6 competes with Edc3 for the interaction with these HLMs. We show that both Edc3 and Scd6 stimulate decapping in vitro, presumably by preventing the Dcp1:Dcp2 complex from adopting an inactive conformation. In addition, we show that the C‐terminal HLMs in Dcp2 are necessary for the localization of the Dcp1:Dcp2 decapping complex to P‐bodies in vivo. Unexpectedly, in contrast to yeast, in metazoans the HLM is found in Dcp1, suggesting that details underlying the regulation of mRNA decapping changed throughout evolution.

HPat provides a link between deadenylation and decapping in metazoa

A proline-rich region in the Drosophila Pat1 homologue works with the proteins C-terminal domain to recruit decapping and deadenylase complexes to target mRNAs.

miRISC recruits decapping factors to miRNA targets to enhance their degradation

MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5′-to-3′ messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5′ cap structure by decapping triggers irreversible decay of the mRNA body. Here, we demonstrate that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation.

A Molecular Link between miRISCs and Deadenylases Provides New Insight into the Mechanism of Gene Silencing by MicroRNAs

MicroRNAs (miRNAs) are a large family of endogenous noncoding RNAs that, together with the Argonaute family of proteins (AGOs), silence the expression of complementary mRNA targets posttranscriptionally. Perfectly complementary targets are cleaved within the base-paired region by catalytically active AGOs. In the case of partially complementary targets, however, AGOs are insufficient for silencing and need to recruit a protein of the GW182 family. GW182 proteins induce translational repression, mRNA deadenylation and exonucleolytic target degradation. Recent work has revealed a direct molecular link between GW182 proteins and cellular deadenylase complexes. These findings shed light on how miRNAs bring about target mRNA degradation and promise to further our understanding of the mechanism of miRNA-mediated repression.