Global analysis of yeast mRNPs.

Proteins regulate gene expression by controlling mRNA biogenesis, localization, translation and decay. Identifying the composition, diversity and function of mRNA-protein complexes (mRNPs) is essential to understanding these processes. In a global survey of Saccharomyces 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 processing bodies (P bodies). Cross-linking and immunoprecipitation (CLIP) 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. When 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.

Scd6 targets eIF4G to repress translation: RGG motif proteins as a class of eIF4G-binding proteins.

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 copurify 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 activators in Saccharomyces cerevisiae act by multiple mechanisms.

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 an 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 preinitiation 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 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.

Crystal structure of human Edc3 and its functional implications.

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 A. 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 site-directed 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.

Structural basis of dcp2 recognition and activation by dcp1.

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.

Crystal structure and functional analysis of Dcp2p from Schizosaccharomyces pombe.

Decapping is a key step in both general and nonsense-mediated 5' --> 3' mRNA-decay pathways. Removal of the cap structure is catalyzed by the Dcp1-Dcp2 complex. The crystal structure of a C-terminally 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.

Crystal structure of Dcp1p and its functional implications in mRNA decapping.

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.