Regulation of mRNA half-life is a crucial control point of gene expression. Removal of the protective 5' methylguanosine cap is a committed step in the 5'-3' decay pathway, which is carried out by the decapping enzyme Dpc2. Although Dcp2 is sufficient for decapping in vitro, its activity is greatly increased by coactivators; here the activation of Dcp2 is explored through several means. A thorough study of the chemistry of decapping is presented: multiple crystal structures implicate specific active site residues of Dcp2 in catalysis metal binding and enzyme kinetics identify key residues involved in the acid/base chemistry of decapping. Further, a metal binding loop is implicated in conformational changes coupled to Dcp2's catalytic cycle using pH-dependent NMR spectroscopy and molecular dynamics simulations. In addition we hypothesized and tested direct effects of other potential regulators of decapping by in vitro assay and binding studies.

In addition, two potential decapping activation pathways are explored. First, a hypothesized interaction between the decapping complex and nonsense-mediated mRNA decay factor Upf1 is explored through GST pull-downs, and enzyme kinetics. Additionally, some evidence suggests the possibility that an extended ribonucleoprotein complex containing mRNA decay factors Pat1/Lsm1-7 and Xrn1 may directly affect the activity of Dcp2; this hypothesis is tested using an in vitro decapping assay. However, future experiments are needed to fully characterize coactivator regulation of Dcp2 within the context of the cell.

Messenger RNA degradation is a fundamental aspect of eukaryotic gene expression, regulation, and quality control. Removal of the 5' N7-methylguanosine (m7G) cap structure by the decapping enzyme Dcp2 is an irreversible step committing an mRNA to degradation by 5'-3' exonucleases. The eukaryotic decapping enzyme is composed of two subunits: Dcp2, which is the catalytic subunit, and Dcp1, and essential activator. Decapping is a tightly regulated process involving a dense network of protein-protein interactions, involving both general and pathway-specific inhibitors of the Dcp1-Dcp2 decapping complex. The purpose of this study is to characterize the interaction between the decapping complex and several coactivators of decapping at the molecular level to begin to understand how coactivators regulate the enzymatic mechanism of Dcp2. Earlier structural studies of Dcp1 and Dcp2 revealed several potential sites where coactivators could bind to the decapping complex. The crystal structure of Dcp1 reveals that it has an EVH1 fold, a protein interaction module that recognizes proline-rich sequences which bind to a surface-exposed pocket of aromatic residues. In this study we used two known enhancers of decapping in Saccharomyces cerevisiae, Edc1 and Edc2, as model coactivators to elucidate the mechanism of activation by proline-rich ligands of Dcp1. Site-directed mutagenesis, fluorescence polarization, and NMR allowed us to characterize the biochemical properties of this enzyme-coactivator system, demonstrating that Edc1 and Edc2 enhance decapping by binding the same site on Dcp1. Using a kinetic assay to monitor mRNA decapping developed in our lab, we determined the kinetic constants of the Dcp1-Dcp2 complex in the presence of coactivators Edc1 and Edc2. We found that Edc1 and Edc2 stimulate decapping by greater than 1000-fold, and that the C-terminus of Edc1 is necessary and sufficient to enhance the catalytic step. This study is the first to demonstrate the importance of the Dcp1 proline-rich sequence (PRS) binding site in the mechanism of decapping. Our findings suggest that proline-rich ligand binding by Dcp1 is an important route utilized by the eukaryotic decapping machinery to recruit substrate and activate Dcp2.

mRNA turnover represents a fundamental point of post-transcriptional control of gene expression in eukaryotes. Decapping is a highly regulated, irreversible step in mRNA decay and is involved in many different decay pathways. The yeast mRNA decapping enzyme is comprised of the catalytic subunit, Dcp2, in complex with Dcp1, an obligate in vivo activator. The goal of this study is to characterize the mechanism of mRNA decapping by Dcp2 and the role of activators.

We embarked on this study by establishing an in vitro model system for decapping. Reconstituting this system required purifying the decapping holoenzyme and the catalytic subunit, preparing radiolabeled RNA substrate and determining optimal assay conditions. This assay allowed us to investigate how Dcp2 binds and recognizes its mRNA substrate, as well as the role of Dcp1 as an activator of decapping, by examining the various kinetic and thermodynamic parameters of this process. We discovered that Dcp2 contains an extensive, highly charged RNA binding surface extending from the active site through a channel to the dorsal side of the protein. This surface is bipartite in function: the dorsal surface binds the RNA body and provides the majority of the binding energy, whereas the cap proximal nucleotides are involved in the catalytic mechanism.

We also determined that Dcp1 stimulates decapping during the catalytic step, wherein Dcp2 undergoes an open-to-closed transition into a catalytically active conformation. This is supported by our result that the cap is recognized during the transition state, suggesting closure promotes cap recognition and enzyme activity. This represents a possible fundamental point of control of this process as activators could shift the balance by promoting the closed, active form of the enzyme. We began studies on one such activator of decapping, Edc3, and discovered that it directly interacts with Dcp2 to activate decapping, though further studies are necessary for determine which step in the catalytic cycle is affected.

This study is the first to mechanistically examine the decapping enzyme, Dcp2, using a multidisciplinary approach involving enzymology, biochemistry, biophysics, and genetics. Our goals were to investigate the structural and biochemical basis for RNA recognition and the mechanism of activation. These studies provide the first insights into control of decapping at the molecular level.

During transcription in the nucleus, messenger RNA (mRNA) is endowed withmodifications that serve as important markers for its regulation in the cell. This includes the addition of a 7-methylguanosine cap (m7G) at the 5’ end, which is important for nuclear export, translation, and degradation of transcripts. Numerous mRNA quality control pathways end in the degradation of transcripts and pathogens can degrade RNA during infection to favor their survival. Removal of the cap structure, or decapping, commits mRNA to degradation and is a highly controlled step in post-transcriptional regulation. The conserved eukaryotic decapping complex Dcp1/Dcp2 is regulated through an intricate network of protein-protein interactions that can inhibit and accelerate decapping. While many of the interfaces required for these interactions have been studied, a mechanistic understanding of their consequences have not been well-characterized. This work describes a mechanism for autoinhibition of decapping and how the protein cofactors Edc3 and Edc1 relieve this autoinhibition to activate decapping through conformational changes in the Dcp2 active site. Furthermore, these regulatory mechanisms contribute to in vitro phase separation that causes localized sites of repressed and accelerated mRNA decapping. Together, these findings demonstrate how regulation at the ångstrom scale is coupled to microscopic behavior to enhance the catalytic power of decapping, which ensures mRNA degradation occurs only when specific conditions are met.

Hijack of the ubiquitin proteasome system is a recurring theme in host-pathogen interactions. HIV-1 encodes at least three `accessory factor' proteins, Vif, Vpr, and Vpu, each known to interact with and retarget a human ubiquitin ligase with deleterious consequences for the host. The best characterized of the accessory factors is Vif, which is required for viral infectivity in T-lymphocytes. In the absence of Vif, the APOBEC3 family of restriction factors is expressed in these cells and induces lethal hypermutation of the viral genome. Vif serves as a viral countermeasure to APOBEC3 proteins by recruiting them to CUL5, a member of the CRL ubiquitin ligases, for K48-linked polyubiquitination thereby sentencing them to proteasomal degradation. Affinity purification-mass spectrometry approaches implicate a large cast of cellular proteins as Vif interactors; the work in this dissertation focuses on understanding the functional significance of a small number of them for Vif. In summary, we find the NEDD8 E2 enzyme UBE2F is critical for activation of the Vif-hijacked ubiquitin ligase, and is recruited to CUL5 by the RING-protein RBX2. The NEDD8 pathway is druggable at the E1 step, and the UBE2F pathway represents a novel drug target for relatively specific inhibition of Vif. Ligands influence the conformational state of Vif, a natively disordered protein. We find that Vif is able to simultaneously bind nucleic acids and components of the ubiquitylation machinery, resulting in a conformation with physical properties not observed in Vif complexes lacking either nucleic acid or ubiquitylation components.

The lifetime of eukaryotic messenger RNA is highly regulated by elements on both the 5' and 3' ends of the message. Methylated guanine "cap" on the 5' end of a message is critical for nuclear export, protein translation, and stability of a message. The cap structure is so crucial that many viruses hijack cellular caps to direct translation towards nefarious ends. Removal of the cap structure of a mRNA is a highly regulated, irreversible step that sentences a mRNA to destruction by conserved exonucleases, and is the ultimate goal of many mRNA surveillance and degradation pathways. An enzyme known as Dcp2 executes decapping, or removal of the cap. Dcp2 from yeast to humans contains two conserved domains: one at the very N-terminus of the protein known as the regulatory domain, and a second following directly in sequence of the Nudix hydrolase family. While the catalytic domain contains the core Nudix motif necessary and sufficient for removal of mRNA caps, the regulatory domain binds the partner Dcp1 and is required for decapping in vivo. The dynamics, structure, and functional consequences of interactions between these two domains are the focus of this manuscript. In sum, regulation of decapping appears to occur by modulating the interaction between these two domains, which creates or inhibits formation of a composite active site that apposes absolutely conserved and catalytically essential regions on both domains.

Bulk RNA degradation irreversibly removes an mRNA from the translating pool to regulate gene expression. Transcripts must be degraded in a coordinated manner during cell cycle, development and in response to stimuli. A dense network of proteins assembles on a transcript to ensure timely and specific destruction of the RNA. This results in trimming of the 3’ poly(A) tail followed by removal of the 5’ methyl-7 guanine (m7G) cap structure, leading to rapid exonucleolysis of the message. A key question has been understanding how protein factors at the 3’ end trigger decapping at the 5’ end. Pat1 is a central scaffolding protein that interacts with multiple decay factors to control distinct steps during RNA turnover. Previous work has demonstrated that Pat1 enhances binding of the heteroheptameric Lsm1-7 complex to the 3’ end and promotes decapping by the Dcp1/Dcp2 enzyme complex at the 5’ end. Due to the multifunctional nature of Pat1, however, we lack a mechanistic understanding of how it regulates RNA turnover. In this work, I have used a reconstituted system to understand how Pat1 interacts with and activates distinct factors during 5’-3’ degradation. First, I show how Pat1 interacts with and enhance the RNA binding of Lsm1-7. This increased affinity is selective for adenine-rich oligoRNAs, which in turn broadens the specificity of the Lsm1-7 complex. Second, I show how Pat1 interacts with short linear motifs in the disordered C-terminal tail of Dcp2 to activate decapping by either recruiting the enzyme complex to substrate or alleviating autoinhibition to promote catalysis. Both activation of Lsm1-7 and decapping require a bipartite interaction between two domains of Pat1 and involve distinct surfaces and motifs. Last, I uncover how different decay factors tune both the size and assembly of Pat1, which may be leveraged to organize an active decapping complex. This biochemically reconstituted system provides a framework for how Pat1 can regulate multiple protein cofactors and steps during bulk RNA turnover.

The decapping complex is a dynamic enzyme that is involved in dynamic processes of development and environment stress responses. In vivo, and to some extent in vitro, decapping has been shown to be modulated by numerous cellular cofactors; including Dhh1 and Pat1:Lsm1-7, which help define the timing of decapping. Additional coactivating sequences contained in lineage specific proteins allow for plasticity in recruiting the complex to novel pathways. Recently, great progress has been made in describing the structured, core domains of the various 5’ decay factors and the decapping structure has been solved in multiple forms that represent various conformations along its enzymatic trajectory; including coactivator bound. Much less is known about the intrinsically disorder regions (IDRs) of these proteins. In vivo results have implicated that there could be a negative regulatory element in the decapping complex. Following this insight, I have discovered and subsequently characterized an autoinhibitory region of the fungal Dcp2 IDR. Additionally, this autoinhibitory complex reveals a potentially larger role for Edc3 in cytoplasmic decay that had previously been unappreciated. This autoinhibition provides new possibilities for our understanding of mRNA decay and this system provides the opportunity to resolve differences between in vivo and in vitro decapping results. Finally, our biochemical and structural insights into the core Dcp1:Dcp2 activation have resulted in an enzyme that may prove useful for RNA cloning methods.