PROJECT SUMMARY Cells organize biochemical reactions into biological condensates. P-bodies are conserved cytoplasmic condensates enriched in factors important for mRNA storage or degradation, but how these opposing outcomes may be achieved in condensates is unclear. A critical step in mRNA degradation is the removal of the 5'-7- methylguanosine cap by the decapping enzyme complex (Dcp1/Dcp2) that precedes and permits digestion of the mRNA body by conserved exoribonucleases. We have reconstituted biological condensates containing fission yeast Dcp1/Dcp2 and an enhancer of decapping protein 3 (Edc3), which are major core proteins of P- bodies. Using novel, activity-based fluorescence probes we have made two significant discoveries. First, contrary to the popular model that condensates enhance enzymatic reactions due to local concentration effects, we find that phase separation represses the activity of Dcp1/Dcp2 100-fold compared to dilute solution. Second, the decapping activity of these condensates can be rescued by Edc3. Our data suggest the protein interaction platform Dcp1 is an integrator of short-linear protein interaction motifs that couples phase separation to inactivation of decapping by promoting a conformational change in Dcp1/Dcp2 to an autoinhibited conformation. In Aim 1, we will determine the structure of the autoinhibited conformation of Dcp1/Dcp2 and test the hypothesis that short-linear motifs in Dcp2 directly interact with Dcp1 to promote a transient inactive conformation of the decapping complex. In Aim 2, we will study how condensates provide an additional layer for decapping repression, testing the hypothesis interactions that promote phase separation further stabilize the inactive conformation of Dcp1/Dcp2 in condensates. In Aim 3, we will determine the mechanism for activation of decapping in condensates, testing the hypothesis that Edc3 opposes the inhibitory action of short-linear inaction motifs in Dcp2 and promotes a conformational change that opens the RNA binding channel in Dcp1/Dcp2 to promote efficient decapping within condensates. Lesions important for repression of decapping in vitro will be tested for their function in EDC3-mediated mRNA decay in fission yeast. The proposed studies are poised to provide a paradigmatic example of how biological condensation is coupled to conformational control of enzyme activity that affects the fidelity of gene expression at the level of mRNA decay.