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.