RNA not only encodes genetic information but also performs a myriad of other biological tasks. This multifunctionality is conferred, in part, by RNA's ability to fold into specific and dynamic three-dimensional structures that often interact with other components of the cell. The diversity of RNA function is particularly useful to viruses, which tend to have genomes that are much smaller than cell-based life and have evolved elegant, subtle, and yet often complex ways to use RNA as part of their infection strategy.
My research focuses primarily on the structure and function of RNA molecules from viruses. Many of these viral RNAs manipulate the host cell's biological machinery through direct intermolecular interactions that are poorly understood. By studying these RNAs and the processes they drive, we learn several things: how the virus operates, how the basic cellular machinery operates, how we might block or inhibit viruses, and the basic rules of RNA and RNA-protein complex structure and function. We explore these systems with a variety of methods, including x-ray crystallography (Figure 1), functional assays, cell-based approaches, biochemistry, and biophysics.
Viral IRES RNAs: Molecular Hijackers
Certain viruses recruit, position, and activate a host cell's ribosomes by a process that does not require the mRNA to be capped, but rather is driven by a structured RNA called an internal ribosome entry site (IRES). IRESs often operate using far fewer protein factors than are needed by the canonical cap-dependent mechanism, and this raises an interesting question: How can RNA structure functionally replace the cap and many protein factors, and in so doing "hijack" the host cell's ribosomes? Using a combination of structural, biochemical, biophysical, and cell-based approaches, we are trying to understand the mechanism of IRESs from viruses as diverse as HIV-1, the hepatitis C virus (HCV), and the intergenic region (IGR) of the Dicistroviridae.
In one project, we are using in vitro and cell-based assays to understand how the 5' leader of the full HIV-1 RNA is involved in translation of viral proteins. Previous reports have identified this leader as a cell-cycle-dependent IRES RNA; we now seek to describe the detailed molecular mechanism that lies behind this observed regulation.
We are also exploring the mechanism by which the HCV IRES is able to manipulate the host cell's translation machinery, with a particular interest in the steps involved in forming 80S ribosomes on the IRES RNA, activating those ribosomes for protein synthesis, and the structural changes in the IRES RNA that accompany these steps. Our strategy is to use functional, biochemical, and structural studies to build a detailed model of HCV IRES function.
In another project, we solved the complete structure of an IGR IRES (Figure 2) and then, using published cryo-electron microscopy (cryo-EM) reconstructions, we proposed a comprehensive structure-based model for the mechanism of this IRES. In this model, the IRES mimics the P/E hybrid-state of a transfer RNA (tRNA), which is the state that tRNAs enter after a peptide bond has been formed but before the tRNAs have translocated on the ribosome. To test this model, we are using methods such as single-molecule fluorescence resonance energy transfer (FRET), and continuing to use biochemical and structural approaches.
Structure and Function of the ciRNA, an Antiviral Countermeasure
Our collaborator, David Barton, recently discovered a sequence in the protein-coding portion of the poliovirus (and other group C enteroviruses) RNA genome that is a competitive inhibitor RNA (ciRNA) of RNase L. RNase L is part of the cell's normal interferon-induced antiviral pathway and hence the ciRNA is an "antiviral countermeasure." We are exploring the three-dimensional structure of the ciRNA and its interactions with the endonuclease domain of RNase L; we are also trying to locate ciRNAs in other viruses (and perhaps cellular organisms). These studies give us insight into how structured viral RNAs can manipulate a host cell's machinery and how a single RNA sequence evolved under two independent pressures: to encode genetic information and to inhibit an RNase.
Multifunctional tRNA-like Structures
Certain economically important plant-infecting RNA virus genomes become aminoacylated at their 3' ends by the host cell's aminoacyl-tRNA synthetases (AARSs) (Figure 3). This reaction depends on RNA sequences, called tRNA-like structures (TLSs), that reside at the 3' end of the genome and that can have sequences and secondary/tertiary structures that differ substantially from authentic tRNAs. TLSs interact with AARSs and other proteins to include the viral RNA-dependent RNA polymerases (RDRPs). It has been proposed that TLSs regulate several steps in the viral replication cycle. This raises two questions: What are the three-dimensional structures and structural dynamics that allow this multifunctionality? How are structurally divergent TLSs able to recruit and manipulate the host cells AARSs? Using biophysical methods such as small-angle x-ray scattering combined with biochemical assays and x-ray crystallography, we are seeking a deeper understanding of these virally encoded molecular mimics, with the long-term goal of understanding how viral RNAs manipulate the cellular machinery (Figure 3).
A long-term goal of RNA structural biologists is to inform the design of small-molecule drugs that target RNA. Attempts to rationally design new drugs that target RNA have not yet succeeded. To inform structure-based drug design efforts, my lab is studying the interactions between small amphiphilic peptides with modified side-chains (designed by Jaehoon Yu) and RNA targets from HIV-1 and HCV. Solving the structures of these peptide-RNA complexes by x-ray crystallography will help us understand how different chemical moieties can interact specifically with RNA. Our goal is to use this information to guide the design of small molecules.
Discovery of New RNA-Protein Complexes in Viral Infections
During infection, viral proteins often bind to cellular RNAs, and viral RNAs can bind to cellular proteins. These interactions are critical to the virus, but in even the best-studied viruses, we have identified probably only a subset of these interactions. To address this gap, we are identifying new RNA-protein complexes that occur during certain viral infections. We are interested in interactions involving cellular proteins and those involving virally encoded proteins. Once identified, our long-term task is to characterize these RNA-protein complexes biochemically, functionally, and biophysically and to use this information to determine how each virus operates in the cell.
Grants from the National Institutes of Health provide additional support for these projects.
As of May 30, 2012