"Illuminating RNA biology with metabolically incorporated ribonucleoside probes"
Host: Eric Kool
About the Seminar
RNA plays a central role in biological processes and characterizing the regulatory mechanisms governing its behavior can reveal fundamental insights into gene expression programs in normal and disease contexts. Towards this end, our lab has developed general approaches based upon RNA metabolic labeling with artificial nucleoside probes in order to study RNA-associated processes transcriptome-wide in live cells and organisms. In the first part, we present RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy using mechanism-based nucleosides that enables characterization of RNA modifying enzymes and their associated post-transcriptional RNA modifications. We apply RNABPP with diverse C5-modified pyrimidine nucleosides in order to identify RNA methyltransferase, dihydrouridine synthase, and dioxygenase enzymes. Further, we combine quantitative RNA mass spectrometry and modification-specific sequencing technologies in order to characterize the abundance and distribution of individual RNA modifications and understand their function in cellular processes. In the second part, we combine protein engineering of the nucleotide salvage pathway with bioorthogonal and fluorescent nucleoside chemistry to develop an approach for live-cell imaging of global RNA dynamics. We apply our method to study RNA trafficking and metabolism upon oxidative stress. Taken together, our work provides powerful and general strategies for interrogating RNA regulation and reveals the presence of novel mechanisms controlling RNA function in biological systems.
About the Speaker
Ralph Kleiner was born in Syracuse, NY and attended Princeton University, where he received an A.B. in Chemistry and performed research on the de novo design of 4-helix bundle proteins in the laboratory of Michael Hecht. He then completed his Ph. D. in Chemistry at Harvard University under the direction of David Liu, where he used DNA-templated chemistry to identify novel macrocyclic inhibitors of Src kinase and Insulin-Degrading Enyzme (IDE). He went on to conduct his postdoctoral studies as a Damon Runyon Postdoctoral Fellow with Tarun Kapoor at The Rockefeller University. His postdoctoral work used chemical proteomics to study phosphorylation-dependent protein-protein interactions in the DNA damage response. In September 2016, Ralph began his appointment as an Assistant Professor in the Department of Chemistry at Princeton University. His research lies on the interface of chemistry and biology, focusing on the development and application of novel chemical approaches to investigate challenging problems in cellular biology. He is particularly interested in understanding how chemical modifications occurring on nucleic acids regulate the function of these macromolecules.
He is a recipient of a Damon Runyon Postdoctoral Fellowship, a Revson Foundation Senior Fellowship in Biomedical Science, the Damon Runyon Dale F. Frey Award for Breakthrough Scientists, the Sidney Kimmel Foundation Scholar Award, the Alfred P. Sloan Foundation Research Fellowship, and the National Science Foundation CAREER Award.