Structural Basis for the Mutually Exclusive Anchoring of P Body Components EDC3 and Tral to the DEAD Box Protein DDX6/Me31B

Tritschler, Felix; Braun, Joerg E.; Eulálio, Ana; Truffault, Vincent; Izaurralde, Elisa; Weichenrieder, Oliver

doi:10.1016/j.molcel.2009.02.014

Cited by 125 publications

(212 citation statements)

References 26 publications

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“…To discriminate between these possibilities, we examined the association of DCP1a with additional components of the decapping complex (i.e., EDC4, DCP2, EDC3, and DDX6/RCK). These all coimmunoprecipitated with DCP1a, as reported before (8,(11)(12)(13)(14) (Fig. 3 A-D, lanes 9).…”

Section: Trimerization Is Required For Dcp1a To Interact With Dcp2 Ansupporting

confidence: 83%

“…Trimerization of DCP1 reveals an unexpected connectivity and complexity of the decapping network in multicellular eukaryotes, as both EDC3 and EDC4 are known or presumed to form homodimers (11,13,15,21). We find that DCP1a can interact with DCP2 and EDC4 independently of the interaction with EDC3 and DDX6/RCK.…”

Section: Dcp1 Trimerization Is Important For Efficient Decapping In Vmentioning

confidence: 71%

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DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa

Tritschler

Braun

Motz

et al. 2009

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

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DCP1 stimulates the decapping enzyme DCP2, which removes the mRNA 5 cap structure committing mRNAs to degradation. In multicellular eukaryotes, DCP1-DCP2 interaction is stabilized by additional proteins, including EDC4. However, most information on DCP2 activation stems from studies in S. cerevisiae, which lacks EDC4. Furthermore, DCP1 orthologs from multicellular eukaryotes have a C-terminal extension, absent in fungi. Here, we show that in metazoa, a conserved DCP1 C-terminal domain drives DCP1 trimerization. Crystal structures of the DCP1-trimerization domain reveal an antiparallel assembly comprised of three kinked ␣-helices. Trimerization is required for DCP1 to be incorporated into active decapping complexes and for efficient mRNA decapping in vivo. Our results reveal an unexpected connectivity and complexity of the mRNA decapping network in multicellular eukaryotes, which likely enhances opportunities for regulating mRNA degradation.DCP2 ͉ miRNAs ͉ P-bodies ͉ EDC4 ͉ Ge-1 I n eukaryotes, removal of the mRNA 5Ј cap structure is catalyzed by the decapping enzyme DCP2 (1, 2); to be fully active and/or stable, DCP2 requires additional proteins (1, 2). Yeast DCP2 interacts directly with DCP1 and this interaction is required for decapping in vivo and in vitro (3-7). In humans, the DCP2-DCP1 interaction requires additional proteins, which together assemble into multimeric decapping complexes that also include the enhancers of decapping 3 and 4 (EDC3 and ECD4), and the DEAD-box protein DDX6/RCK (8, 9).DCP2 is highly conserved and most information on DCP2 activation stems mainly from studies in S. cerevisiae and S. pombe (3-7). Fungi, however, lack EDC4 as well as many extensions and additional domains present in decapping activators of metazoan orthologs (8-10). For example, all eukaryotic DCP1 proteins contain an N-terminal EVH1 domain (3, 5, 6); however, DCP1 orthologs from metazoa and plants also have a proline-rich C-terminal extension (9, 10). The sequence of this extension is not conserved except for a 14-residue short motif (motif I, MI) conserved in metazoa with the exception of C. elegans (Fig. 1A and Fig. S1) and a C-terminal domain conserved in plants and metazoa ( Fig. 1 A, referred to as TD).The DCP1 C-terminal domain is predicted to adopt an ␣-helical conformation. In this work, we show that this domain trimerizes in an asymmetric fashion. We solved the crystal structure of the trimerized domain for both human and D. melanogaster DCP1 and show that the trimer adopts an unprecedented fold, with no current similarities in the protein database. We further show that DCP1 trimerization is required for the assembly of active decapping complexes and for mRNA decapping in vivo. The conservation of structurally critical residues indicates that this domain adopts a similar fold in DCP1 orthologs of other multicellular eukaryotes. Consequently, within mRNA decapping complexes in these organisms, the stoichiometry of the protein components is likely more complex than previously thought. Results and DiscussionCry...

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Section: Trimerization Is Required For Dcp1a To Interact With Dcp2 Ansupporting

confidence: 83%

Section: Dcp1 Trimerization Is Important For Efficient Decapping In Vmentioning

confidence: 71%

DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa

Tritschler

Braun

Motz

et al. 2009

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

Self Cite

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“…We also tested a mutant with substitutions on the FDF‐binding surface of DDX6 (Mut2, Appendix Table S1). These mutations prevent the interaction with 4E‐T and decapping factors, which all bind to the FDF‐binding surface of DDX6 in a mutually exclusive manner (Fig 8A–D; Tritschler et al , 2009; Ozgur et al , 2015). However, DDX6 Mut2 still binds to NOT1 (Fig 8E), indicating that the mutations do not alter the protein fold.…”

Section: Resultsmentioning

confidence: 99%

mi RISC and the CCR 4– NOT complex silence mRNA targets independently of 43S ribosomal scanning

et al. 2016

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AbstractmiRNAs associate with Argonaute (AGO) proteins to silence the expression of mRNA targets by inhibiting translation and promoting deadenylation, decapping, and mRNA degradation. A current model for silencing suggests that AGOs mediate these effects through the sequential recruitment of GW182 proteins, the CCR4–NOT deadenylase complex and the translational repressor and decapping activator DDX6. An alternative model posits that AGOs repress translation by interfering with eIF4A function during 43S ribosomal scanning and that this mechanism is independent of GW182 and the CCR4–NOT complex in Drosophila melanogaster. Here, we show that miRNAs, AGOs, GW182, the CCR4–NOT complex, and DDX6/Me31B repress and degrade polyadenylated mRNA targets that are translated via scanning‐independent mechanisms in both human and Dm cells. This and additional observations indicate a common mechanism used by these proteins and miRNAs to mediate silencing. This mechanism does not require eIF4A function during ribosomal scanning.

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“…It is also remarkable that Gle1 and eIF4G, two very different proteins, both activate two distinct DEAD box proteins using a similar structural and mechanistic basis. A part of the sites that these regulators bind seems to be a hotspot for association with DEAD box helicases, as suggested by studies of DDX6 bound to a fragment of its regulator enhancer of mRNA-decapping protein 3 (EDC3) 125 , and of eIF4AIII binding to its regulato r UPF3B 126 .…”

Section: Nuclear Specklesmentioning

confidence: 99%

From unwinding to clamping — the DEAD box RNA helicase family

Linder

Jankowsky

2011

Nat Rev Mol Cell Biol

983

1,069

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Nearly all aspects of RNA metabolism, from transcription and translation to mRNA decay, involve RNA helicases, which are enzymes that use ATP to bind or remodel RNA and RNA-protein complexes (ribonucleoprotein (RNP) complexes) 1 . RNA helicases are found in all three domains of life, and many viruses also encode one or more of these proteins 2,3 . Together with the structurally related DNA helicases that function in replication, recombination and repair, the RNA helicases are classified into superfamilies and families, based on sequence and structural features 3,4 . DEAD box proteins form the largest helicase family, with 37 members in humans and 26 in Saccharomyces cerevisiae 3 , and are characterized by the presence of an Asp-Glu-Ala-Asp (DEAD) motif.DEAD box helicases have central and, in many cases, essential physiological roles in cellular RNA metabolism 5 (FIG. 1). The proteins generally function as part of larger multicomponent assemblies, such as the spliceosom e or the eukaryotic translation initiation machinery 6 . Mutations and deregulation of several DEAD box proteins have been linked to disease states, including cancer 7 . Although all DEAD box proteins contain a structurally highly conserved core with conserved ATP-binding and RNA-binding sites, different proteins have been associated with diverse and seemingly unrelated functions, including the disassembly of RNPs, chaperoning during RNA folding and even stabil ization of protein complexes on RNA 5,6 . How these highly conserved proteins fulfil such an array of different functions has been a long-standing question.Research has now started to illuminate the molecular basis for this functional diversity. In this Review, we summarize how structural data, together with biochemical and biophysical studies, have revealed unexpected modes by which these proteins function. We also discuss how novel molecular and cell biological approaches have better defined the cellular roles of DEAD box helicases. We outline our current view on common structural and mechan istic themes that have emerged, and on physical models of how these 'unusual' RNA helicases work.Structures of DEAD box RNA helicases A number of structural studies have universally shown that members of the DEAD box family contain a highly conserved helicase core that harbours the binding sites for ATP and RNA 3,4,8 . The core is surrounded by variable auxiliary domains, which are thought to be critical for the diverse functions of these enzymes.Structure of the helicase core. DEAD box proteins belong to helicase superfamily 2 (SF2) 2 . Similarly to all SF2 helicases, DEAD box proteins are built around a highly conserved helicase core of two virtually identical domains that resemble the bacterial recombination protein recombinase A (RecA) 3,4,8 (FIG. 2a,b). Within this helicase core, at least 12 characteristic sequence motifs are located at conserved positions (FIG. 2a,b). Some of these motifs are conserved across the entire SF2 family, whereas others are found only in the DEAD box family 3 ....

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Structural Basis for the Mutually Exclusive Anchoring of P Body Components EDC3 and Tral to the DEAD Box Protein DDX6/Me31B

Cited by 125 publications

References 26 publications

DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa

DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa

mi RISC and the CCR 4– NOT complex silence mRNA targets independently of 43S ribosomal scanning

From unwinding to clamping — the DEAD box RNA helicase family

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