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Pat1 is a hub for mRNA metabolism, acting in pre-mRNA splicing, translation repression, and mRNA decay. A critical step in all 5′-3′ mRNA decay pathways is removal of the 5′ cap structure, which precedes and permits digestion of the RNA body by conserved exonucleases. During bulk 5′-3′ decay, the Pat1/Lsm1-7 complex engages mRNA at the 3′ end and promotes hydrolysis of the cap structure by Dcp1/Dcp2 at the 5′ end through an unknown mechanism. We reconstitute Pat1 with 5′ and 3′ decay factors and show how it activates multiple steps in late mRNA decay. First, we find that Pat1 stabilizes binding of the Lsm1-7 complex to RNA using two conserved short-linear interaction motifs. Second, Pat1 directly activates decapping by binding elements in the disordered C-terminal extension of Dcp2, alleviating autoinhibition and promoting substrate binding. Our results uncover the molecular mechanism of how separate domains of Pat1 coordinate the assembly and activation of a decapping messenger ribonucleoprotein (mRNP) that promotes 5′-3′ mRNA degradation.

The conserved decapping enzyme Dcp2 recognizes and removes the 5′ eukaryotic cap from mRNA transcripts in a critical step of many cellular RNA decay pathways. Dcp2 is a dynamic enzyme that functions in concert with the essential activator Dcp1 and a diverse set of coactivators to selectively and efficiently decap target mRNAs in the cell. Here we present a 2.84 Å crystal structure of K . lactis Dcp1–Dcp2 in complex with coactivators Edc1 and Edc3, and with substrate analog bound to the Dcp2 active site. Our structure shows how Dcp2 recognizes cap substrate in the catalytically active conformation of the enzyme, and how coactivator Edc1 forms a three-way interface that bridges the domains of Dcp2 to consolidate the active conformation. Kinetic data reveal Dcp2 has selectivity for the first transcribed nucleotide during the catalytic step. The heterotetrameric Edc1–Dcp1–Dcp2–Edc3 structure shows how coactivators Edc1 and Edc3 can act simultaneously to activate decapping catalysis. The decapping enzyme Dcp2 removes the 5′ eukaryotic cap from mRNA transcripts and acts in concert with its essential activator Dcp1 and various coactivators. Here the authors present the structure of the fully-activated mRNA decapping complex, which reveals how Dcp2 recognizes the cap substrate and coactivators Edc1 and Edc3 activate catalysis.

Cells organize biochemical processes into biological condensates. P-bodies are cytoplasmic condensates that are enriched in enzymes important for mRNA degradation and have been identified as sites of both storage and decay. How these opposing outcomes can be achieved in condensates remains unresolved. mRNA decapping immediately precedes degradation, and the Dcp1/Dcp2 decapping complex is enriched in P-bodies. Here, we show that Dcp1/Dcp2 activity is modulated in condensates and depends on the interactions promoting phase separation. We find that Dcp1/Dcp2 phase separation stabilizes an inactive conformation in Dcp2 to inhibit decapping. The activator Edc3 causes a conformational change in Dcp2 and rewires the protein–protein interactions to stimulate decapping in condensates. Disruption of the inactive conformation dysregulates decapping in condensates. Our results indicate that the regulation of enzymatic activity in condensates relies on a coupling across length scales ranging from microns to ångstroms. We propose that this regulatory mechanism may control the functional state of P-bodies and related phase-separated compartments. Alterations in the interactions driving phase separation of the mRNA decapping complex led to conformational rearrangements in its active site, providing a mechanism to control whether substrate mRNA is stored or decapped in condensates.

Poxviruses encode decapping enzymes that remove the protective 5' cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral dsRNA, a trigger for antiviral responses. Here we present the first high resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a novel domain organization in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5' mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap binding proteins VP39, eIF4E, and CBP20 and distinct from eukaryotic decapping enzyme Dcp2. Competing Interest Statement The authors have declared no competing interest.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.

Pat1 is a hub for mRNA metabolism, acting in pre-mRNA splicing, translation repression and mRNA decay. A critical step in all 5′-3′ mRNA decay pathways is removal of the 5′ cap structure, which precedes and permits digestion of the RNA body by conserved exonucleases. During bulk 5′-3′ decay, the Pat1/Lsm1-7 complex engages mRNA at the 3′ end and promotes hydrolysis of the cap structure by Dcp1/Dcp2 at the 5′ end through an unknown mechanism. We reconstitute Pat1 with 5′ and 3′ decay factors and show how it activates multiple steps in late mRNA decay. First, we find that Pat1 stabilizes binding of the Lsm1-7 complex to RNA using two conserved short-linear interaction motifs. Secondly, Pat1 directly activates decapping by binding elements in the disordered C-terminal extension of Dcp2, alleviating autoinhibition and promoting substrate binding. Our results uncover the molecular mechanism of how separate domains of Pat1 coordinate the assembly and activation of a decapping mRNP that promotes 5′-3′ mRNA degradation.

Cells organize biochemical processes into biological condensates. P-bodies are cytoplasmic condensates enriched in factors important for mRNA degradation. P-bodies have been identified as sites of both mRNA storage and decay, but how these opposing outcomes may be achieved in condensates is unresolved. A critical step in mRNA degradation is removal of the 5’-7-methylguanosine cap by Dcp1/Dcp2, which is highly enriched in P-bodies. Dcp1/Dcp2 activity is repressed in condensates in vitro and requires the activator Edc3. Activation of decapping is amplified in condensates relative to the surrounding solution due to stabilization of an autoinhibited state in Dcp1/Dcp2. Edc3 couples a conformational change in the Dcp1/Dcp2 active site with alteration of the protein-protein interactions driving phase separation to activate decapping in condensates. The composition-dependent regulation of enzyme activity in condensates occurs over length scales ranging from microns to Ångstroms and may control the functional state of P-bodies and related phase-separated compartments. mRNA decapping in droplets is repressed Catalytically inert droplets are activated by a change in condensate composition A switch in enzymatic activity requires a conformational change in condensates Condensates amplify enzyme activation compared to surrounding solution

5' mediated cytoplasmic RNA decay is a conserved cellular process in eukaryotes. While the functions of the structured core domains in this pathway are understood, the role of abundant intrinsically disordered regions (IDRs) is lacking. Here we reconstitute the Dcp1:Dcp2 complex containing a portion of the disordered C-terminus and show its activity is autoinhibited by linear interaction motifs. Enhancers of decapping (Edc) 1 and 3 cooperate to activate decapping by different mechanisms: Edc3 alleviates autoinhibition by binding IDRs and destabilizing an inactive form of the enzyme, whereas Edc1 stabilizes the transition state for catalysis. Both activators are required to fully stimulate an autoinhibited Dcp1:Dcp2 as Edc1 alone cannot overcome the decrease in activity attributed to the C-terminal extension. Our data provide a mechanistic framework for combinatorial control of decapping by protein cofactors, a principle that is likely conserved in multiple 5' mRNA decay pathways.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary time scales. Building on our previous work (Mavor et al. 2016), we used deep mutational scanning to determine how twelve new chemicals reveal novel mutational sensitivities of ubiquitin residues. We found sensitization of Lys63 in eight new conditions. In total, our experiments have uncovered a highly sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the Ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.