Project Summary: RNA is perhaps the most versatile molecule in the cell due to its significant contribution to nearly every cellular process. But we still don’t really understand many aspects of how RNA features, such as nucleotide modifications and secondary and tertiary structure, work in concert to regulate its many functionalities. The central dogma of biology tells us that DNA is transcribed to RNA, which then is translated to protein. We already know that DNA has a complex web of structural organization and covalent modification which provides transcriptional control. But modifications, splicing, and structure of RNA involve an even more complex interplay to regulate protein production. Yet without appropriate tools, we cannot explore this world. Here we propose to leverage the newly developed direct RNA nanopore sequencing platform to directly interrogate RNA, enabling us to examine modifications to RNA and their interplay with the dynamics of splicing and RNA structure. Nanopore sequencing directly probes the chemical structure of the molecule in the pore, allowing us to measure endogenous RNA modifications; but we first need to understand the raw electrical signal data produced, through the generation of training sets. To explore the dynamics of RNA production and splicing, we will utilize this toolbox to examine non-canonical nucleotides, e.g. 4sU, that we inject as a metabolic label for nascent RNA. For structural probing, we will adapt conventional techniques which label unpaired RNA nucleotides to allow high-throughput examination of complex RNA structure. These tools and analysis pipelines will grant new insights into mechanisms of transcriptional regulation, as well as their implications for human disease.