Research & Projects

Research in the Gross Lab at UC San Francisco focuses on understanding the molecular-level details of multi-protein complexes that bind RNA or DNA and are important for the control of human gene expression and disease. Proteins coordinate and carry out all of the diverse biochemical functions of a cell. Few proteins act in isolation; instead, they interact with many different protein co-factors and RNA or DNA to form dynamic, multi-protein molecular machines. These multi-protein complexes—which can quickly assemble, disassemble, or change composition—allow the cell to finely tune and regulate biochemical processes. But their large sizes and complexities make them difficult to study in high detail using traditional approaches.

Regulation of RNA decay by protein-protein interactions

The regulation of eukaryotic mRNA plays a crucial role in many biological processes such as development, stress response, and gene expression. One major pathway used to regulate cellular mRNA levels is 5’-to-3’ mRNA decay. A critical step in this pathway requires removal of the protective 5’-terminal N7-methyl-guanosine (m7G) cap found on all eukaryotic mRNA, which commits the transcript to degradation by conserved 5’-to-3’ exonucleases. Hydrolysis of the m7G cap is catalyzed by the decapping enzyme Dcp2, in complex with the essential activator Dcp1. Dcp2 is a bilobed, Nudix hydrolase that is conserved from yeast to humans and interacts with a variety of coactivators in addition to Dcp1 (Edc1-3, Pat1/Lsm1-7, and Dhh1 in yeast, for example) and mRNA to form a messenger ribonucleoprotein that carries out the decapping reaction. Dcp2 is essential for microRNA-mediated degradation of mRNA transcripts in Drosophila and important for degradation of long non-coding RNAs in yeast. These two classes of non-coding RNAs are important for the maintenance of cellular equilibrium in mammals and abnormal levels of micro or long non-coding RNAs are found in many human cancers. Despite the biological importance of decapping and 5’-to-3’ mRNA decay, the structural details of mRNA cap cleavage by Dcp2 and its activation by protein-protein interactions with coactivators remain poorly understood.

RNA Lifecycle

RNA life cycle overview, highlighting decapping mRNP and 5’-to-3’ mRNA decay.

In the Gross Lab, we are developing a molecular-level understanding of how Dcp2 catalyzes mRNA cap cleavage and how the decapping reaction is activated and regulated by protein-protein interactions with coactivators, in order to explain the structural basis of mRNA decapping in cells.
DCP Open Closed

Conformational equilibrium of the Dcp1/2 decapping complex. Work from our lab has shown that the Dcp1/2 complex is dynamic, with open-to-closed transitions of the two domains of Dcp2 occurring on the us-ms timescale. Dcp1 is yellow, Dcp2 N-terminal regulatory domain is purple, and Dcp2 catalytic domain is green. Structures are from the Song lab (cite: She 2008, PDB 2QKM).

We combine a broad selection of experimental techniques that include NMR spectroscopy, X-ray crystallography, small-angle X-ray scattering, fluorescence anisotropy, in vitro decapping kinetics, and yeast genetics, in order to understand the links between the structure and conformation of the decapping complex with its cellular function. Recent work in the Gross Lab has shown that the conserved N-terminal regulatory domain of Dcp2 and the activator Dcp1 stimulate catalysis of m7G cap cleavage by the catalytic domain of Dcp2 by ~100 fold and ~10 fold in vitro, respectively (Deshmukh Mol Cell 2008 and Floor NSMB 2010). Addition of the coactivator Edc1, which binds a conserved surface of Dcp1, stimulates Dcp2-mediated decapping by an additional ~1,000 fold (Borja RNA 2010). Using a combination of NMR and computational studies, we have shown that Dcp2 is a dynamic enzyme that undergoes both global (open-to-closed) and local (active site residues) conformational changes, which are essential for catalysis and may couple coactivator binding to stimulation of decapping (Floor PNAS 2012; Aglietti Structure 2013). We propose a model for mRNA decapping catalysis by Dcp2 in which the regulatory and catalytic domains of Dcp2 come together to form a closed, bipartite active site that recognizes and cleaves the m7G cap structure (Floor RNA Biology 2008). Coactivator proteins may accelerate cap hydrolysis by binding the Dcp1/2 decapping complex and further promoting the closed, catalytically active conformation of the enzyme.

DCP 12 Mechanism

Proposed catalytic cycle for decapping of mRNA by Dcp1/2 and coactivator Edc1. The two domains of Dcp2 form a bipartite active site that recognizes and cleaves m7G cap (red square). Edc1 accelerates catalysis by binding to Dcp1 and promoting the closed, catalytically active conformation of the enzyme.

Structural biology of HIV-host protein complexes

Many viral pathogens co-evolve with host organisms to persist in spite of host immune defenses. As part of the HARC Center at UCSF, our lab is studying how viral HIV proteins interact with human host proteins to cause infection and disease. The APOBEC3 family of restriction factors (A3D, F, G, and H) is a potent arm of the primate innate immune system that restricts HIV infection via hypermutation of viral genome intermediates formed during HIV replication. To counter the antiviral APOBEC3 proteins, HIV-1 (and nearly all other lentiviruses) encodes the accessory protein Viral infectivity factor (Vif). The primary function of Vif is to hijack a cellular Cullin-RING ubiquitin ligase and target APOBEC3 proteins for degradation by the 26S proteasome.

In the Gross Lab, we are deconstructing the intermolecular interactions between viral immunomodulatory proteins and host proteins at the molecular level to improve our understanding of both host immunity and viral pathogenesis, and to aid in the development of new classes of antiviral therapeutics.

We combine a broad selection of structural and biophysical techniques to probe how Vif hijacks the host ubiquitin ligase complex and confers species-specific recognition of APOBEC3 proteins. In a collaborative effort with other members of the HARC Center at UCSF, we have identified a novel Vif interaction partner, the cellular transcription cofactor CBF-β, which is required for the proper function of the Vif E3 ubiquitin ligase (cite/link: Jager Nature 2011). The Gross Lab has recently made breakthroughs in biochemical reconstitution and structural studies of the Vif E3 ubiquitin ligase (Kim Mol Cell 2013). Using in vitro ubiquitiation assays, we determined that our reconstituted Vif E3 ligases are enzymatically active and recapitulate the well-established properties of Vif observed in cells. Furthermore, we demonstrated that pharmacological inhibition of Vif function unleashes the restriction potential of APOBEC3 family members, and suggest Vif as a promising therapeutic target for innovative drug development (Stanley PLoS Pathog 2012).

HIV Overview