PROJECT SUMMARY Throughout biology, RNA molecules form complex and dynamic networks of molecular interactions that are essential to their function, but which remain challenging to investigate. These networks of RNA-interacting proteins, RNAs, and genomic loci regulate nearly all aspects of mRNA function, enable noncoding RNAs that regulate gene expression at various levels, and scaffold molecular assemblies that control cellular gene- expression, metabolic, and stress-response programs. Dysregulation of RNA-interactions has been causally implicated in numerous human pathologies, suggesting that these interactions may represent a significant class of untapped therapeutic targets. Yet, despite the central importance of RNA to basic biology and human disease, methods for elucidating the factors that interact with any given RNA remain limited. Current state-of- the-art approaches—which use biotinylated antisense oligonucleotides to pull down target RNAs from crude cell lysates—are noisy, suffer from low target RNA specificity, and lack biological context. Emerging strategies that use transgenically expressed enzymes to affinity-tag RNA-interactors in situ require complicated cell-line engineering that limits their applicability across cell types and target RNAs. Therefore, there is a pressing need for straightforward and generalizable tools that can elucidate intra-cellular RNA-interactions at high resolution, without cumbersome biochemical fractionation or cell-line engineering. To meet this challenge, and in response to RFA PAR 19-253, this proposal will develop Oligonucleotide-Directed Biotinylation (ODB). This novel technique combines high-resolution single-molecule RNA-FISH and in situ proximity-biotinylation to map RNA interaction networks within their native cellular context. In pilot experiments, ODB exhibited exceptionally precise targeting of individual RNAs in situ, and enabled proteomic analysis of RNA-scaffolded structures that are difficult to isolate biochemically. We have also recently demonstrated that proximity-biotinylation approaches like ODB can be used to probe nucleic acids as well as proteins. Given these promising proof-of- principle results, we propose developing ODB into a unified, “multi-‘omic” method for identifying the proteins, RNAs, and/or genomic loci that interact with a broad range of target RNAs. In Aim 1, we will optimize the core steps of the ODB workflow, developing robust protocols for deploying ODB to a target RNA at high spatial precision, and for isolating RNA-interacting proteins, RNAs, and genomic loci from an ODB experiment. We will develop general-use strategies for applying ODB in an array of different mammalian cell lines and RNA targets. In Aim 2, we will “field test” ODB on a dynamic, developmentally-regulated nuclear-architectural RNA that has been difficult to characterize by conventional approaches. These experiments will develop a versatile and straightforward technology for interrogating RNA interactions in situ, and which is easily adoptable by most laboratories. Given the pervasive roles played by RNA throughout biology, this transformative method will pave the way for paradigm-shifting discoveries in cell biology, and reveal novel RNA-based therapeutic targets.

PROJECT NARRATIVE Although RNAs drive a diverse array of cellular functions throughout biology, and are causally dysregulated in many human pathologies, probing the interactions RNA molecules make within cells remains technically challenging. To address this technological gap, in this proposal we will develop a robust and straightforward method for identifying the proteins, RNAs, and genomic loci that interact with a target RNA, that can be easily applied to different RNAs and cellular states. This technology will transform our understanding of RNA interactions across numerous biological settings and diseases, paving the way for paradigm-shifting discoveries in cell biology, and revealing novel RNA-based therapeutic targets.