Research summary:
RNA lies at the very center of life as we know it, and continued discoveries of new RNA functions lend credence to the idea that it is the most versatile of the biological macromolecules. RNA can encode and decode genetic information, perform catalysis, sense metabolite concentrations, control gene expression, initiate translation, and perform many other tasks critical for life. In addition, it is an essential part of many cellular machines and structures, such as ribosomes, spliceosomes, signal recognition particles, telomerases, etc. RNA is truly multifunctional and this characteristic is a direct result of its ability to form specific, dynamic, and diverse three-dimensional structures.
In the Kieft Lab, we want to understand the fundamental features of RNA that allow this “multifunctionality,” while also contributing to knowledge that can benefit human health. Viruses with RNA genomes cause human illnesses such as chronic hepatitis, AIDS, cancer, influenza, ebola, and polio, and other RNA viruses have become severe agricultural problems, such as foot-and-mouth-disease virus, the Dicistroviruses (contributing to crashing bee populations and widespread shrimp death), and various plant viruses. Hence, we are focusing our efforts on RNAs that are found in viruses and that perform interesting and important functions.
Our overarching goal is to understand how the function of an RNA molecule results from its structure. In order to do this, we use a variety of methods and approaches that we combine to yield a cohesive picture. For example, we do not just seek to solve the structure of an RNA that we are interested in, but also to understand how different parts of that structure contribute to the function, how the structure changes as it functions, what interacts with the RNA, and how mutations affect structure and function. Methods we employ include traditional biochemical approaches (e.g. chemical probing, pull-downs, binding assays), biophysical methods (e.g. ultracentrifugation, SAXS), structural methods (e.g. X-ray crystallography, NMR), and functional assays (e.g. translation assays, aminoacylation assays, ribosome assembly assays). Specific projects in the Kieft Lab include:
Non-protein coding IRES RNAs from hepatitis C virus (HCV), HIV-1, and the Dicistroviridae
Internal ribosome entry site (IRES) RNAs drive initiation of protein synthesis by a pathway that is very different from the pathway used by the majority of cellular mRNAs. Viruses use these IRES RNAs to “hijack” the host cell’s protein making machinery and thus make viral proteins critical for infection (Figure 1). IRES RNAs have been identified as potential drug targets, but before we can exploit that idea we need to know much more about their structure and how that structure drives function. In the Kieft Lab, we are studying three diverse IRESs to learn about the different ways IRESs work, and also find any common mechanistic “rules” that all use. Our approach to understanding these RNAs is driven by the specific questions we are asking, rather than by our desire to use a certain technique. Hence, we are using many methods, including X-ray crystallography, NMR spectroscopy, RNA/protein pull-downs combined with Mass Spec., translation assays (in vitro and in cell culture), chemical and enzymatic probing, ribosome assembly assays, and ultracentifugation. In addition, we are beginning cryo-EM and single-molecule FRET studies in collaboration with other labs in an attempt to bring the most powerful emerging techniques to bear on these problems.
In the last few years, we have succeeded in solving the first complete structure of an IRES RNA (intergenic region from the Dicistroviridae) (Figure 1). These structures are not the end of our research on this RNA; rather they allow us to develop new testable models and design the next set of experiments to understand dynamics and function. In addition, the ideas we have developed based on this IRES have allowed us to look at the HCV IRES and the HIV-1 IRES in new ways, and to design experiments to understand these IRESs to a greater level of detail.
Peptide inhibitors of viral RNAs
Our work on these IRESs aims to find new drug targets, with special emphasis on those parts of the RNA that dynamically change structure during the mechanism of translation initiation. We have found several promising targets and to exploit these, we recently entered into a collaboration to design small peptides with modified side-chains in an attempt to find inhibitors of these IRES RNAs (and other viral RNAs). Initial studies have identified several promising lead peptides, and we are now trying to understand how these peptides bind to the RNA and alter its structure, and then use this information to guide small drug design efforts.
RNase-inhibiting RNA (ciRNA) from poliovirus.
Ribonuclease L (RNase L) is activated as part of the interferon-dependent antiviral response, efficiently cleaving RNAs. RNase L is also linked to regulation of cell proliferation, apoptosis, translation regulation, leukemia, Type I diabetes, chronic fatigue syndrome, and hereditary prostate cancer. Recently, our collaborator, Prof. David Barton (University of Colorado School of Medicine) discovered a highly conserved sequence in the coding region of poliovirus RNA that is a potent competitive inhibitor of RNase L, acting by binding directly to the protein’s endonuclease domain (Figure 2). This novel RNA is the first example of a naturally occurring RNA that inhibits an RNase.
To understand this RNA, we are employing crystallography, RNA-protein binding assays, mutagenesis, modification interference, heteronuclear NMR, small-angle X-ray scattering, and RNA structure-based bioinformatics efforts to find analogous RNAs in other systems. We have already shown that this RNA assumes a compact fold that includes a long-range “kissing hairpin” interaction. Preliminary crystallization trials show that it is crystallizable, as we have RNA-only crystals that diffract to ~7 Å and will pursue RNA-protein complex crystals. The goal is to understand in depth how this RNA binds to the enzyme, and also to understand how binding blocks function. We anticipate that our studies will reveal how this novel RNA functions, and also address the larger question of how structured RNAs can interact with and change the function of other molecules in unexpected ways.
tRNA-like structures from plant viruses
Certain plant viral genomic RNAs contain sequences at their 3' end that structurally mimic tRNAs, being aminoacylated by host cell synthetases (Figure 3). We are interested in understanding this mimicry in depth, as these RNA must not only be recognized by the host cell’s synthetases, but also by elongation factors, and RNA polymerases. Hence, these RNAs appear to be multifunctional platforms that might coordinate several steps in the viral replication cycle, and may dynamically change structure as they do so. The presence of these RNAs also raises interesting questions about how prevalent tRNA mimicry might be. Our preliminary investigation of these RNA reveals that the presumption of absolute tRNA structural mimicry may be overstated; some of these RNA do not appear to have stable folds while some have folds that appear to deviate dramatically from tRNA. What does this say about the evolution of RNA structure in three-dimensional space? How precise must RNA mimicry be in order to manipulate other biological machines? We are addressing these questions, again using a combination of biochemical, structural, and biophysical methods. It will be interesting to see if our investigation into these potential tRNA mimics forges a link back to the mimicry observed in RNAs such as tmRNA, which is involved in the rescue of stalled ribosomes in bacteria.
The phi29 phage DNA packaging motor
More than a decade ago it was discovered that bacteriophage j29 encodes a non-protein coding RNA (dubbed “pRNA”) that is an integral and necessary part of the ATP-dependent “nanomotor” that packages DNA into the maturing phage prohead. The self-assembling motor consists of 12 copies of phage protein gp10, and multiple copies of protein gp16 and the pRNA. The pRNAs form intermolecular base-pairs (called “hand-in-hand” interactions) in a closed ring structure that can form without proteins. Some evidence shows this ring is hexameric, other reports show it is pentameric, and some hypothesize the number changes during motor assembly. This is far from an academic question, as the mechanism of the motor remains unclear and the number of copies of pRNA in the motor profoundly affects how it might work. Could this self-assembling RNA-containing nanomotor be exploited as a DNA or drug delivery machine? As a first step, we must understand how the motor works, and a key part of this is determining the role of the pRNA, its stoichiometry, and its interactions with gp10 and gp16. A large body of biochemical and biophysical data already exists, as does a crystal structure of the gp10 dodecamer ring and cryo-EM reconstructions of the prohead with the motor bound. But the field is fundamentally limited by the lack of complete high-resolution structural information. Therefore, we are undertaking the ambitious goal of solving the crystal structures of the isolated pRNA ring, the pRNA-gp10 complex and the fully assembled gp10-gp16-pRNA motor. We believe that having a full suite of structures is the best way to understand how the machine assembles and how it works.
Representative Publications:
Selected recent publications from the Kieft Lab
Hammond. J.A., Rambo, R.P., Filbin. M.E.,& Kieft, J.S. (2008). Comparison and functional implications of the 3-D architectures of viral tRNA-like structures. RNA (in press).
Costantino, D.A., Pfingsten, J.S., Rambo, R.P., & Kieft. J.S. (2008) tRNA-mRNA mimicry drives translation initiation from a viral IRES. Nat. Struct. Mol. Biol.15, 57-64.
Keel, A.Y., Rambo. R.P., Batey, R.T., & Kieft, J.S. (2007) A general method to solve the phase problem in RNA crystallography. Structure 15, 761-772.
*Batey, R.T. & *Kieft, J.S. Improved native affinity purification of RNA (2007) RNA, 13, 1384-1389.
*These authors contributed equally to this work
Pfingsten, J.S., Costantino, D.A & Kieft, J.S. (2007) Conservation and diversity among the three-dimensional folds of the Dicistroviridae intergenic region IRESes. J. Mol. Biol., 370, 356-369.
Pfingsten, J.S., Costantino, D.A., & Kieft, J.S. (2006) Structural basis for ribosome recruitment and manipulation by a viral IRES. Science, 314, 1450-1454.
Costantino, D. & Kieft, J.S. (2005). A preformed compact ribosome-binding domain in the cricket paralysis-like virus IRES RNAs. RNA 11, 332-343.
*Kieft, J.S. & *Batey, R.T. (2004). A general method for rapid and nondenaturing purification of RNAs. RNA 10, 988-995.
*These authors contributed equally to this work
Recent reviews articles and book chapters from the Kieft Lab
Kieft, J.S. Biophysical analysis of IRES RNAs from the Dicistroviridae: linking architecture to function. Springer Series in Biophysics, invited book chapter (in press).
Kieft, J.S. (2008) Comparing the three-dimensional structures of Dicistroviridae IGR IRES RNAs with other viral RNA structures. Virus Res. Epub ahead of print, PMID: 18672012 (Coeditor of this edition)
Kieft, J.S. (2008) Viral IRES RNA structures and ribosome interactions. TiBS, 33, 274-283.
Pfingsten, J.S. & Kieft, J.S. (2008) RNA structure-based translation initiation: Lessons from the Dicistroviridae intergenic region IRESs. RNA, 14, 1255-63.
Kieft, J.S., Costantino, D.A., Filbin, M.E., Hammond J., and Pfingsten, J.S. (2007) Structural methods for studying IRES function. Methods in Enzymology, 430, 333-371.
Kieft, J.S. & Pfingsten, J. (2005) Weapons in the molecular arms race (News and Views). Nat. Struct. Mol. Biol. 12, 938-939.
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