Ralph Kleiner

CHEMICAL APPROACHES TO STUDY POST-TRANSCRIPTIONAL RNA REGULATION

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, the Kleiner lab has developed approaches based upon the manipulation of cellular nucleic acid chemistry in order to study post-transcriptional RNA regulation through RNA modifications and RNA-binding proteins. In the first part, we present RNA-mediated activity-based protein profiling (RNABPP), a chemoproteomic strategy using mechanism-based modified nucleoside probes 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 toidentify 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 describe the development and application of a small-molecule dependent RNA editing strategy to study cellular RNA-protein interactions and their involvement in biomolecular condensate structures. Taken together, our work provides powerful strategies for interrogating RNA regulation and reveals the presence of novel mechanisms controlling RNA function in biological systems.