Manipulating and Interrogating Spatial Transcriptomics

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ABSTRACT Spatial mRNA organization plays a fundamental role in diverse cellular processes and disease. In large, compartmentalized cells (e.g., neurons and embryos), subcellular mRNA localization offers a core mechanism for the spatiotemporal regulation of protein synthesis. Since the initial discovery of subcellular mRNA distribution in 1983, high-throughput imaging and sequencing methods have revealed that, in many cell types, thousands of RNAs are localized to distinct compartments. For example, many axonal-related mRNAs in neurons will transport to the “site of needed” along the very long (>100μm) axon, which likely play an important role in axon development and local synaptic activities. Furthermore, mounting evidence shows a correlation between aberrant spatial RNA organization and an increasing number of diseases, including amyotrophic lateral sclerosis (ALS), fragile X syndrome (FXS), and spinal muscular atrophy (SMA). However, due to a lack of technologies that allow for the tracking and manipulation of the spatial localization of endogenous mRNAs in primary cells and in vivo, the mechanism and functional relevance of spatial organization has only been explored for a small number of mRNAs. In this proposal, we seek to establish a set of technologies as a new foundation to study spatial RNA biology, by developing an integrated framework that allows for sophisticated computational analysis, real-time RNA tracking, and programmable spatial manipulation of any endogenous mRNA(s) in situ and in vivo, on a high-throughput (>1,000 mRNAs in parallel) scale. To achieve this goal, we will start by building a deep learning framework that can analyze spatially localized RNAs in different cell types and predict their associated regulatory factors (e.g., RNA motifs, RNA binding proteins). This will provide an atlas of spatial RNA organization as well as candidate RNAs for functional studies. Next, we will develop two novel approaches, RNA live-cell fluorescent in situ hybridization (RNA-LiveFISH) for single-molecule, real-time dynamic tracking, and CRISPR- mediated transcript organization (CRISPR-TO) for programmable manipulation of any target mRNA localization. The two approaches form a new framework that enables us to study the regulatory mechanism and functional relevance of subcellular mRNA localization with unprecedented ease and spatiotemporal resolution. Third, we seek to apply this framework to study the function of mRNA localization in primary neurons, via high-throughput manipulation of >1,000 mRNAs to uncover functions for axon guidance, growth cone development, and synaptic activities. Selected functional mRNAs (>100) will be verified in vivo. Finally, we will apply the framework to investigate the pathological mechanisms of aberrant RNA localization underlying the neurological disease spinal muscular atrophy (SMA) in vitro and in vivo. We will not only dissect the relationship between mRNA organization and SMA pathology, but also explore the strategy of modulating RNA localization for potential therapeutics. We envision that the proposal will lead to new groundbreaking insights into the mechanism and functional roles of whole-cell mRNA spatial organization for cellular and physiological functions that has not been revealed before.