Regulation of mRNA decapping by protein-protein interactions

mRNA decay is highly regulated and plays a critical role in early animal development, cell growth and proliferation, adaptation to stress, and the quality control of gene expression. The 5'-3' decay pathway initiated by decapping is intimately tied to many of these mRNA degredation systems. Various protein factors involved in homeostatic mechanisms as well as specific decay pathways such as nonsense-mediated decay have been shown to promote decapping of substrates. How is this accomplished?

Recent work has shown that a conformational change is required for catalysis by the mRNA decapping enzyme, Dcp2. We have shown that the conserved N-terminal domain of Dcp2 and the activator Dcp1 stimulate catalysis by the catalytic domain of Dcp2 by ~1,000 fold yet only transiently associate with the catalytic domain in solution by NMR (Deshmukh, et al. 2008). A crystal structure from the Song lab shows the Dcp1/Dcp2 complex in two conformations: one extended and inactive (open) and the other bound to nucleotide and compact (closed) (She, et al. 2008). The open and closed forms of the crystal structure are depicted below. The activator and protein-protein interaction platform Dcp1 is shown in yellow with the N-terminal and catalytic Nudix domains of Dcp2 in blue and green, respectively.

The aforementioned studies have provided a model of catalysis by Dcp2 which proposes that the above conformational change is used as a point of control by decapping activators. This model is illustrated below. Dcp2 exists in a conformational equilibrium between open and closed forms (a). RNA binds to the open form (b). After binding the enzyme closes over the RNA substrate (c). This is the step that co-activators may enhance. Following closure the m7G cap is hydrolyzed (d). The RNA is then released (e) and subject to exonucleolysis by 5'-3' nucleases such as Xrn1 and Rat1.

We seek to understand the genetic, biophysical, and mechanistic consequences of interactions with co-activators of decapping in bulk decay as well as specific surveillance pathways such as nonsense-mediated decay. Crystallography has provided invaluable insight into the mechanism of Dcp2 but critical questions regarding conformational dynamics and their role in catalysis and decapping stimulation remain unanswered. NMR is uniquely suited to give per-residue and global dynamical information on fast (ns-ps) to slow (ms) motions. We are using NMR in concert with in vitro kinetic experiments, SAXS, and yeast genetics to understand the purpose of the open-closed transition in Dcp2, the role of a coupling between global and local dynamics, and the step in catalysis at which decapping activators work.

Structural Biology of HIV·Host complexes

We are also investigating how the HIV protein Vif disarms a human nucleic acid immune system that recognizes and hypermutates viral genome intermediates formed during HIV replication. The human proteins APOBEC3G and APOBEC3F convert cytosine to uracil in newly reverse-transcribed HIV cDNA, thereby causing viral hypermutation and severely restricted viral replication. In turn, Vif acts as a countermeasure by recruiting APOBEC3G and 3F to a cellular Ubiquitin E3 ligase consisting of Cullin-5, Rbx2, and Elongins B and C. This complex catalyzes formation of polyubiquitin chains on APOBEC3G and 3F, targeting them for destruction by the 26S proteosome. In collaboration with labs at the HARC center we are using a variety of biophysical, biochemical and cell biological methods to understand the structure and function of the Vif E3 ligase. The ultimate goal of these studies is to provide a new route for the treatment of HIV by developing small molecular inhibitors that inhibit assembly of the Vif E3 ligase.

NMR is used to investigate transient macromolecular interactions , to explore allostery and provide de novo structures of domains or subcomplexes that are refractory to crystallization. The NMR facility at UCSF enables the research carried out by participating HARC laboratories and outside collaborators. For example, structural studies of APOBEC3G are performed at UCSF as part of a HARC center collaboration with the Matsuo laboratory at University of Minnesota. Laboratories within the HARC center are using NMR to investigate secondary binding sites within the Rev response element and to examine interactions between Nef and host factors.