Eukaryotic mRNA degradation often occurs in a process whereby translation initiation is inhibited and the mRNA is targeted for decapping. In yeast cells, Pat1, Scd6, Edc3, and Dhh1 all function to promote decapping by unknown mechanism(s). We demonstrate that purified Scd6 and a region of Pat1 directly repress translation in vitro by limiting the formation of a stable 48S pre-initiation complex. Moreover, while Pat1, Edc3, Dhh1 and Scd6 all bind the decapping enzyme, only Pat1 and Edc3 enhance its activity. We also identify numerous direct interactions between Pat1, Dcp1, Dcp2, Dhh1, Scd6, Edc3, Xrn1, and the Lsm1-7 complex. These observations identify three classes of decapping activators that function either to directly repress translation initiation and/or stimulate Dcp1/2. Moreover, Pat1 is identified as critical in mRNA decay by first inhibiting translation initiation, then serving as a scaffold to recruit components of the decapping complex, and finally activating Dcp2.

Proteins regulate gene expression by controlling mRNA biogenesis, localization, translation and decay. Identifying the composition, diversity and function of mRNPs (mRNA protein complexes) is essential to understanding these processes. In a global survey of S. cerevisiae mRNA binding proteins we identified 120 proteins that cross-link to mRNA, including 66 new mRNA binding proteins. These include kinases, RNA modification enzymes, metabolic enzymes, and tRNA and rRNA metabolism factors. These proteins show dynamic subcellular localization during stress, including assembly into stress granules and P-bodies (Processing-bodies). CLIP (cross-linking and immunoprecipitation) analyses of the P-body components Pat1, Lsm1, Dhh1 and Sbp1 identified sites of interaction on specific mRNAs revealing positional binding preferences and co-assembly preferences. Taken together, this work defines the major yeast mRNP proteins, reveals widespread changes in their subcellular location during stress, and begins to define assembly rules for P-body mRNPs.

A critical step in mRNA degradation is the removal of the 5' cap structure, which is catalyzed by the Dcp1-Dcp2 complex. The crystal structure of an S. pombe Dcp1p-Dcp2n complex combined with small-angle X-ray scattering analysis (SAXS) reveals that Dcp2p exists in open and closed conformations, with the closed complex being, or closely resembling, the catalytically more active form. This suggests that a conformational change between these open and closed complexes might control decapping. A bipartite RNA-binding channel containing the catalytic site and Box B motif is identified with a bound ATP located in the catalytic pocket in the closed complex, suggesting possible interactions that facilitate substrate binding. Dcp1 stimulates the activity of Dcp2 by promoting and/or stabilizing the closed complex. Notably, the interface of Dcp1 and Dcp2 is not fully conserved, explaining why the Dcp1-Dcp2 interaction in higher eukaryotes requires an additional factor.

The formation of mRNPs controls the interaction of the translation and degradation machinery with individual mRNAs. The yeast Scd6 protein and its orthologs regulate translation and mRNA degradation in yeast, C. elegans, D. melanogaster, and humans by an unknown mechanism. We demonstrate that Scd6 represses translation by binding the eIF4G subunit of eIF4F in a manner dependent on its RGG domain, thereby forming an mRNP repressed for translation initiation. Strikingly, several other RGG domain-containing proteins in yeast co-purify with eIF4E/G and we demonstrate that two such proteins, Npl3 and Sbp1, also directly bind eIF4G and repress translation in a manner dependent on their RGG-motifs. These observations identify the mechanism of Scd6 function through its RGG-motif and indicate that eIF4G plays an important role as a scaffolding protein for the recruitment of translation repressors.

Decapping is a key step in both general and nonsense-mediated 5′ → ′ mRNA-decay pathways. Removal of the cap structure is catalyzed by the Dcp1-Dcp2 complex. The crystal structure of a Cterminally truncated Schizosaccharomyces pombe Dcp2p reveals two distinct domains: an all-helical N-terminal domain and a C-terminal domain that is a classic Nudix fold. The C-terminal domain of both Saccharomyces cerevisiae and S. pombe Dcp2p proteins is sufficient for decapping activity, although the N-terminal domain can affect the efficiency of Dcp2p function. The binding of Dcp2p to Dcp1p is mediated by a conserved surface on its N-terminal domain, and the N-terminal domain is required for Dcp1p to stimulate Dcp2p activity. The flexible nature of the N-terminal domain relative to the C-terminal domain suggests that Dcp1p binding to Dcp2p may regulate Dcp2p activity through conformational changes of the two domains. mRNA degradation has an important role in post-transcriptional regulation of gene expression. Decapping is a crucial control point in the life of eukaryotic mRNAs, as it permits the degradation of the transcript and is a site of numerous control inputs 1 . For example, transcripts with premature translation termination codons can be degraded by rapid deadenylationindependent decapping 2 in a process referred to as nonsense-mediated decay 3 . In addition, decapping may be important in the AU-rich element (ARE)-mediated decay pathway in mammalian cells, where ARE-binding proteins are thought to recruit the decapping complex and other mRNA-decay enzymes to ARE-containing mRNAs 4,5 . Finally, recent results indicate that the RNA-mediated interference machinery interacts with the decapping enzyme and decapping might have a role in the reduction of mRNA levels by microRNAs 6,7 .The decapping enzyme complex is composed of Dcp2p (the catalytic subunit) and Dcp1p 8 . Recombinant yeast and human Dcp2 proteins have been shown to have decapping activity 9-11 , with human Dcp2 being a more robust enzyme than its yeast counterpart. Moreover, Dcp2p physically interacts with Dcp1p in human, S. cerevisiae and S. pombe 9,11-13 , and genetic experiments in yeast indicate that Dcp2p requires Dcp1p to work as a holoenzyme in vivo 14, 15 . Dcp1p is a small protein containing an EVH1 domain, a protein-protein interaction module

A major pathway of eukaryotic mRNA turnover begins with deadenylation, followed by decapping and 5′→3′ exonucleolytic degradation. A critical step in this pathway is decapping, which is carried out by an enzyme composed of Dcp1p and Dcp2p. The crystal structure of Dcp1p shows that it markedly resembles the EVH1 family of protein domains. Comparison of the proline-rich sequence (PRS)-binding sites in this family of proteins with Dcp1p indicates that it belongs to a novel class of EVH1 domains. Mapping of the sequence conservation on the molecular surface of Dcp1p reveals two prominent sites. One of these is required for the function of the Dcp1p-Dcp2p complex, and the other, corresponding to the PRS-binding site of EVH1 domains, is probably a binding site for decapping regulatory proteins. Moreover, a conserved hydrophobic patch is shown to be critical for decapping.The process of mRNA turnover is important in numerous aspects of eukaryotic mRNA physiology. These roles include the control of gene expression, antiviral defenses 1,2 and mRNA surveillance by recognizing and degrading the aberrant mRNAs 3 . Two major pathways of mRNA decay exist in eukaryotic cells 4 . In both yeast and mammals, mRNA degradation usually begins with the shortening of the poly(A) tail at the 3′ end of the mRNA. After deadenylation, the 5′ cap structure can be removed (decapping), thereby exposing the transcript to digestion by a 5′→3′ exonuclease Xrn1p 5-8 . Alternatively, after deadenylation in both yeast and mammals, mRNAs can be degraded in a 3′→5′ direction by the cytoplasmic exosome 9-12 . The resulting cap structure is hydrolyzed by the DcpS scavenger decapping enzyme, which is encoded by the DCS1 gene in yeast 13 .Decapping is a key step in the 5′→3′ decay pathway because it induces degradation of the mRNA, and thus it is subject to numerous control inputs. Decapping is also critical in an aspect of mRNA surveillance in which aberrant mRNAs containing nonsense codons are decapped without a requirement for deadenylation and are degraded in a 5′→3′ direction [14][15][16] RESULTS Overall structure of Dcp1pThe crystal structure of Dcp1p was solved by single wavelength anomalous dispersion (SAD) using selenomethionine (SeMet)-substituted crystals. The final model contains two copies of the Dcp1p, designated as molecules A and B, respectively. Several regions of the polypeptide are not visible in the electron density map and are assumed to be disordered, namely residues 1-21, 74-134 and 229-231 for molecule A, and residues 1-16 and 81-134 for molecule B in the crystallographic asymmetric unit. The current refined model has an R-factor of 22.7% and a R free value of 26.8% at a resolution of 2.3 Å with very good stereochemistry (see Table 1 and Methods). m 7 GDP could not be located in the electron density map or in washed crystals, suggesting that m 7 GDP probably does not bind in the crystal lattice (data not shown).No substantial differences are observed between the structures of the two Dcp1p molecules in the asymmetric uni...

Edc3 is an enhancer of decapping and serves as a scaffold that aggregates mRNA ribonucleoproteins together for P-body formation. Edc3 forms a network of interactions with the components of the mRNA decapping machinery and has a modular domain architecture consisting of an N-terminal Lsm domain, a central FDF domain, and a C-terminal YjeF-N domain. We have determined the crystal structure of the N-terminally truncated human Edc3 at a resolution of 2.2 Å. The structure reveals that the YjeF-N domain of Edc3 possesses a divergent Rossmann fold topology that forms a dimer, which is supported by sedimentation velocity and sedimentation equilibrium analysis in solution. The dimerization interface of Edc3 is highly conserved in eukaryotes despite the overall low sequence homology across species. Structure-based sitedirected mutagenesis revealed dimerization is required for efficient RNA binding, P-body formation, and likely for regulating the yeast Rps28B mRNA as well, suggesting that the dimeric form of Edc3 is a structural and functional unit in mRNA degradation.The controlled turnover of eukaryotic mRNA is crucial for regulating gene expression (11,25,29,42). Two general pathways exist to degrade eukaryotic mRNAs, both of which are initiated by deadenylation (23,35,39). Following deadenylation, mRNAs can be degraded 3Ј to 5Ј by the cytoplasmic exosome or, more commonly, are decapped by the Dcp1/Dcp2 decapping enzyme and degraded by the 5Ј-to-3Ј exonuclease, Xrn1p. The formation of an mRNA ribonucleoprotein (mRNP) capable of decapping correlates with the mRNA ceasing translation and forming a translationally repressed mRNA (13, 38). These translationally repressed mRNPs can aggregate in the cytoplasm to form processing bodies (P-bodies), which are dynamic cytoplasmic RNA granules. P-bodies are of interest, as the mRNPs within them have been implicated in translation repression (13, 26), general mRNA decay (14, 44), nonsense-mediated mRNA decay (45, 49), microRNA-mediated translational repression (32, 41), and mRNA storage (5, 6).An important aspect of understanding the process of mRNA decapping and P-body formation is to understand the interactions and functions of the proteins that form the translationally repressed mRNP capable of decapping and P-body localization. A variety of genetic, biochemical, cell biology, and genomic analyses have indicated that the mRNP capable of decapping and P-body localization contains a variety of conserved proteins that interact with each other and RNA, including the decapping enzyme Dcp1p/Dcp2p, the decapping activators Dhh1p, Lsm1-7p, Pat1p, and Edc3p, the 5Ј-3Ј exoribonuclease Xrn1p, and the Ccr4p-Pop2-Not1p deadenylase complex (20,38). It has also been shown that P-body formation can be affected by the pool of nontranslating mRNAs (20,38) and that proteins such as RCK/p54/Dhh1 and Pat1p contribute to P-body formation by increasing the pool of nontranslating mRNAs (13, 40). Recently, Dcp2p was found to contribute to P-body formation, presumably through its multiple interactions wi...

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