PROJECT SUMMARY Cellular RNAs have more than 100 different kinds of chemical modifications, which together comprise the epitranscriptome. Previous studies have revealed that RNA-modifying pathways are vital to higher eukaryotes, and the alteration of these pathways is associated with a number of human diseases. However, it is still unclear why we need such a complex chemical repertoire for RNA and how RNA modifications impact gene expression in biological tissues with diverse cell types. The major bottleneck for investigating RNA modifications has been the lack of measurement tools with subcellular and single-cell resolutions. Bulk epitranscriptomic sequencing methods can only measure the averaged distribution of modification sites from millions of cells, obscuring the intrinsic heterogeneity of epitranscriptomic states in distinct cell types. Moreover, spatial information is lost during RNA extraction from tissues, preventing further analysis of RNA modification patterns in the context of tissue morphology and subcellular compartments. To address such limitations, we propose to create cutting-edge platforms for three-dimensional (3D) in situ sequencing of RNA modifications. Then we will utilize the new tools to analyze gene regulation mechanisms mediated by RNA modifications at single-cell resolution in intact biological tissues. Using the N6-methyladenosine (m6A) pathway in mouse brain tissue as our model system, we will (a) develop innovative and broadly applicable 3D in situ sequencing methods for RNA modifications; (b) profile the m6A code at single-cell resolution within intact mouse brain tissue; (c) study how m6A modifications in various cell types work collectively and interactively to regulate brain function. In total, this proposal will lead to (1) a transformative toolbox for single-cell epitranscriptomic profiling with broad applications in various biological tissues, (2) a spatially resolved single-cell RNA modification atlas in the mouse brain, and (3) new scientific understanding on how RNA modifications impact tissue function. In the long term, we aim to reveal new principles of post-transcriptional gene regulation at subcellular and single-cell resolutions in complex biological systems, as well as discover new chemical fingerprints of RNAs in health and disease.

PROJECT NARRATIVE RNA (the transcriptome) is central in the flow of biological information from DNA to protein, while chemical modifications of RNA (the epitranscriptome) represent an additional layer of regulatory information to control cell state and fate. Existing methods can only profile mRNA modifications from mixtures of millions of cells, limiting our ability to understand mRNA modifications in tissues that are comprised of diverse cell types in various states. We propose to develop a new set of tools to enable transcriptome-wide profiling of mRNA modifications with single-cell and spatial resolutions, and further apply these new tools to reveal whether different cell types regulate RNA modifications differently and how gene regulation events in single cells collectively give rise to tissue function and dysfunction.