Research in our group focuses on characterizing the structure, dynamics, and function of ribonucleic acids (RNAs) involved in fundamental cellular processes such as catalysis and regulation of gene expression by implementation of classical and novel NMR methodologies. We also venture into other biophysical tool such as SAXS, X-ray crystallography, as well as computational methods, NMR pulse-sequence development and chemical biology technologies to aid in our quest for answers to interesting biological questions.
Messenger RNA splicing is a fundamental process in message maturation to ensure translation of the appropriate protein product in eukaryotic cells. Its failure,misplicing, is implicated in a range of human diseases (i.e. Alzheimer’s, cystic fibrosis and various cancers). In order to better understand the inner workings of misplicing, the mechanism of splicing has to be understood at the molecular level. To perform this task, we are investigating the structural biology of a model system, the group II intron, to identify its catalytic mechanism and its key molecular players.
Gene Expression Regulation
Gene regulation was long thought to be a purely protein-driven process. However, this past decade it was proven that RNA alone plays also a major role in regulation of gene expression at both the transcriptional and translational level. For instance, many cases have been observed where the 5’ untranslated region of an mRNA acts as a sensor of small metabolites producing a change in the RNA’s conformation that could up- or down-regulate expression of the gene downstream. In our lab, we investigate a series of these riboswitches and other regulatory RNA elements to unveil the structural and dynamic basis for their ability to regulate gene activity.
RNA-Drug Interactions
Aminoglycosides and macrolides are known to interact with ribosomal RNA to exert their antibiotic effects. As in these two examples and with increasing evidence of RNA as gene regulators, efforts at new drug discovery and design are focusing not only at targeting proteins and DNAs but also RNAs. Some act within macromolecular complexes such as the ribosome, while others likely act within smaller regulatory components such as riboswitches. Therefore, a major effort underway in our lab is screening for various drugs and drug targets that fall within these categories using structure assisted approaches. Later, these RNA-drug complexes could be potentially useful in pre-clinical studies.
Research Project Description
NMR structural biologists are faced with a few challenges when studying macromolecules even after overcoming the isotope enrichment the problem since the most naturally abundant isotopes of carbon and nitrogen are not NMR-active. For example, once a uniformly 13C and 15N enriched RNA sample reaches the threshold of 30 nucleotides or longer, signal broadening and severe chemical shift overlap become significant to the extent that NMR peak assignments becomes nearly impossible or even observation of any peaks is troublesome as they become vanishingly small. Our lab is therefore devoted to developing technologies to overcome these obstacles.
Biomass Method: The use of bacterial metabolic pathways to obtain amino acids containing stable 13C or 15N isotopes in specific locations has been well established. In our laboratory, we utilize the same principles of following the flow of different metabolites through the various metabolic pathways to ultimately engineer site-specifically labeled nucleotides. Moreover, by careful examination of these pathways, we now utilize mutant bacterial strains that allow for more customized labeling patterns. Once the nucleotides are harvested and processed accordingly, they are ready for use in in vitro transcription of any RNA of interest.
Chemoenzymatic synthesis: Sometimes, the isotope labeling pattern we desire is not achievable by the Biomass method. We therefore resort to an in vitro technique for synthesizing isotopically labeled nucleotides. With starting materials such as ribose and a purine/pyrimidine bases, we can quantitatively synthesize any nucleotide by utilizing a set of enzymes from the pentose phosphate pathway. The newly made nucleotides are readily purified and utilized for in vitro transcription of any RNA of interest. In our lab we are developing the technology that will allow any experimenter to custom make any set of nucleotides to cater to any experimental need.
Native RNA preparation: It has been established that, just as for proteins, RNA can also undergo an intricate folding process, which ultimately determines whether the biomolecule is functional or not. Also, it is thought that in vitro transcription and refolding-folding processes used for the traditional purification of RNAs may not adequately recover the native fold of an RNA understudy, especially for larger RNAs (>30nt).Therefore, to circumvent this problem, our lab is devising techniques to prepare RNAs that keep their native fold from beginning to end utilizing the bacterial folding machinery in the process. Additionally, this technique can also be coupled to both biomass and chemoenzymatic isotopic labeling technologies to obtain site-specifically labeled natively folded RNA.
Research Project Description
Pulse-sequence development: In the past, development of new labeling technologies changed the field of protein NMR; we anticipate that novel site specific labeling technologies being developed by our group as described above will also change the landscape of RNA NMR by providing opportunities to develop new NMR experiments that make use of these new labels, especially for large RNA molecules. These will include experiments for resonance assignments, for extraction of parameters for modeling the 3D structures of RNAs, for monitoring the interactions of RNAs with ligands such as drugs or other RNAs, as well as for more accurate measurements of relaxation parameters.