RNA is a remarkably versatile biological macromolecule, performing tasks ranging from encoding genetic information, performing catalysis, forming the scaffolds for large macromolecular machines, manipulating enzymes, binding small molecules, etc. It is safe to say that the RNA World is alive and well.
How does RNA perform so many biological tasks? The answer lies, in part, in the ability of RNA to form diverse three-dimensional structures whose complexity rivals that of proteins. Thus, to fully understand how RNA operates in healthy and diseased cells, we need to understand the structures of RNA in many ways: how structures form, their conformational dynamics, their stability, what they interact with, etc. But is not enough to just see structures, we need to use this structural information to understand the fundamental basis for RNA function and how it is dictated and regulated by structure.
The strategy of the RNA Kieft Lab has been to focus on exploring RNAs of viral origin. It is estimated that there are 1037 viral particles on Earth at any moment; these represent a rapidly evolving, structurally and functionally diverse treasure-trove of interesting and novel RNAs to explore. Because viruses are obligate cellular parasites, their RNAs are rapidly evolving new ways to interact with and manipulate the cellular biological machinery. Thus, by studying these RNAs we learn fundamental things about basic biological processes and how RNA operates in cells. What is found in viral RNAs is often later found to be true of cellular RNAs. Finally, viruses remain substantial human health threats and a detailed molecular understanding of how they manipulate their cellular hosts is required to develop new therapeutics.
Our goal is to understand these RNAs and their function in detail. We are not satisfied to know that that a certain mutation in a specific RNA causes a loss of function, we want to know why. We ask questions such as: What is the architecture and the conformational dynamics of a certain folded RNA? What does the RNA interact with and how? What are the implications of these interactions? Do mutations alter the structure of the RNA? How? What does this mean in terms of function? Our approaches include cell culture-based methods with disease relevant cells, in vitro activity assays, biochemical studies, x-ray crystallography, biophysical methods, and virology.
A long-term interest of the lab are viral internal ribosome entry sites (IRESs). Certain eukaryote-infecting viruses recruit, position, and activate a host cell’s ribosomes by a process that does not require the 5’ end or the 5’ cap, but rather is driven by a structured RNA called an IRES. IRESs often operate using far fewer protein factors than are needed by the canonical cap-dependent mechanism, and this raises interesting questions: How can RNA structure functionally replace the cap and many protein factors, and in so doing “hijack” the host cell’s ribosomes? How do IRES operate in changing cellular conditions, and how can a class of IRESs be “fine-tuned” to work in diverse cell types? What are the detailed interactions between IRESs and the translation machinery and how does this allow them to manipulate the machinery? We are using a variety of biochemical assays, cell-based assays, and cryo-electron microscopy to answer these questions.
For example, some plant-infecting RNA viruses have a 'tRNA-like structure' (TLS) at the 3’ end of their genome that is aminoacylated, and this is important for success of the virus. Another example is the 'vtRNAs' that are produced during gammaherpes virus infection, exported to the cytoplasm, packaged into the viral particle, but whose function remains mysterious.
Some IRES RNAs can also mimic tRNAs, and almost certainly there are other tRNA mimics waiting to be discovered. What are the structures of these RNAs? What do they interact with? How tRNA-like are they? We are fascinated by the idea that there may be undiscovered biological pathways in both healthy and diseased cells that use tRNA-like molecules. Currently, we are using a variety of biochemical, structural, and functional methods to study tRNA-like RNAs, understand their structures, and relate this to function.
Our collaborator, Dr. David Barton, 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 this remarkable RNA, the first RNA found to inhibit an RNase. We want to understand its interactions with the endonuclease domain of RNase L and how this blocks enzymatic activity. We are also developing cell-based infection models to understand the effect of these RNA structures on the success of the virus. These studies give us insight into how structured viral RNAs can manipulate a host cell’s machinery, and also how a single RNA sequence evolved under two independent pressures: to encode genetic information and to inhibit an enzyme.
Mosquito-borne flaviviruses (FVs) include West Nile Virus, Yellow Fever Virus, Dengue Virus, and Zika Virus. During infection by these viruses, a small flaviviral RNA (sfRNA) of ~300-500 nt accumulates. This sfRNA is essential for viral replication and cytopathicity and thus understanding how it is made is critical for understanding the virus. Remarkably, this sfRNA is produced by co-opting the host cell’s canonical mRNA degradation machinery, specifically by incomplete degradation of the viral genomic RNA by cellular exonuclease Xrn1. We call the RNA elements capable of this 'Xrn1 resistant RNAs' (xrRNAs).
We are fascinated by this process and are exploring the mechanical, thermodynamic, and structural features that allow an RNA to resist an enzyme evolved to degrade it. We have solved the crystal structure of two examples. Now, we are thinking more about how these Xrn1 resistant elements and the sfRNAs they form are involved in infection, and how patterns of sfRNAs are generated in different cell types lead to different outcomes. We are also exploring the ability of xrRNAs from more diverse viruses (both flaviviruses and others) to block exonucleases and are now searching for other examples of xrRNA throughout the virological and biological. We hypothesize that exonuclease resistance may be a novel maturation mechanism for producing smaller RNAs from larger RNAs in an elegant and programmed way. This area is the subject of several collaborations.
An important component to our research is the constant desire to improve and integrate existing tools to study RNA (particuarly RNA structure). We are also interested in using what we learn of these viral RNAs, or RNA structure in general, to create new tools that may be useful to a wider research community. Current efforts include exploring how Xrn1-resistant RNAs can be used to control RNA degradation in cells, the design of RNAs with novel folds for nanotechnology applications (with the Lab of Rhiju Das at Stanford), and the use of RNAs as targets for small-molecule or within mRNA-based therapies (with industry partners).