University of Colorado Department of Biochemistry and Molecular Genetics

Kieft Lab research interests - overview

RNA is the most versatile of all biological macromolecules. It can encode genetic information (like DNA) and also fold into complex structures essential for complicated biological functions (like proteins). Research in the Kieft Lab is seeks to understanding how the structures of viral RNA drive diverse function, motivated by the fact that: 1) Viral RNAs have evolved to perform myriad functions, often of amazing subtly and elegance, giving us a diverse set of RNAs to study. 2) Viruses remain a substantial human health threat and a detailed molecular understanding of them is required to develop new therapeutics. 3) Many viral RNAs manipulate the host cell’s machinery; by studying how this happens, we learn fundamental things about basic biological processes.

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 like analytical ultracentrifugation and thermal denaturation, chemical probing, native gel electrophoresis, mass spectrometry, etc. We are also beginning to employ single-molecule FRET experiments (in collaboration with the lab of Ruben Gonzalez, Columbia Univ.) and hope to soon start cryo-EM studies.

Viral IRES RNAs: molecular hijackers. Of major interest in our lab and the focus of many student thesis projects are viral internal ribosome entry sites (IRESs). 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 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. Current efforts include crystallographic studies of IRES RNA domains bound to ribosomes, smFRET studies of IRES-ribosome complexes, identification of factors that bind to IRESs, and mapping of the functional regions of IRES RNAs.

tRNA-like structures: mimics of ancient molecules. Transfer RNAs (tRNAs) are ancient and fundamentally critical for life. It is not surprising that some viruses mimic tRNAs in diverse ways. 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 various tRNA-like RNAs from diverse sources, understand their structures, and relate this to function.

Poliovirus ciRNA: an elegant antiviral countermeasure. 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. 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.

Development of new methods. An important component to our research is the development of new methods for RNA structural studies. Recently, we developed a new method to purify structural quantities of RNAs using an affinity-tag based system and also a method to incorporate heavy atoms into RNA structures, allowing "first-time, every-time" phasing of RNA crystal diffraction data (collaboration with R. Batey, UC Boulder). These techniques have the potential to change the way RNA structural studies are conducted. We continue to test new methods in the lab, including HPLC-based methods for purification of RNA and large RNA-containing complexes, capillary electrophoresis-based RNA structural analysis, and new ways to study large RNPs…stay tuned.