The human body comprises of trillion of cells, which are the building blocks of life. Each cell is highly organized, with various constituents within localizing to specific regions. Location within a cell is a cardinal organizing principle of gene control and function, especially for RNAs that code for proteins or serve regulatory roles as non-coding RNAs. Each cell type has its distinct RNA-species profile, and where these RNAs are located within the cell can dictate their folding, editing, splicing, translation, degradation, binding partners, catalytic activity, and even the fate of the proteins that they encode. Unsurprisingly, perturbations in RNA localization result in disease conditions including cancers and neuropathologies. In spite of the vital importance of subcellular RNA localization in gene expression, investigating such phenomena simultaneously for many RNA species has been challenging. To address these long-standing issues, Dr. Furqan Fazal and others have recently developed a technique called APEX-seq that can track the location of thousands of RNAs in living human cells with high-temporal and nanometer-spatial resolution. APEX-seq has the transformative potential to provide high-resolution snapshots of tens of thousands of endogenous cellular RNA species, free from artifacts of overexpression, cell fixation or recombinant tagging. This proposal outlines a five-year career program for Dr. Fazal as he investigates principles of RNA subcellular localization, and reaches milestone goals that will transition him into an independent investigator. By improving the APEX-seq technique and amalgamating it with a number of cutting-edge tools including massively-parallel reporter assays and deep-learning-based approaches, Dr. Fazal will dissect how sequences within RNAs direct them to different locations, and how cells actively control RNA subcellular localization to regulate gene function. This work will be carried out at Stanford University, where Dr. Fazal will be mentored by Dr. Howard Chang, a pioneer in developing new genomics tools, and Dr. Joanna Wysocka, a leading authority in vertebrate development and non-coding genomic regulatory elements. Both mentors have highly-successful track records of placing postdoctoral fellows into independent academic positions at leading institutions around the nation and world. Additional support will be available through collaboration with Drs. Shen, Boettiger and Kundaje who will provide training and expertise in genetics, imaging, statistics and computation. An advisory committee comprising of world-leading investigators Drs. Ting, Kool, Li, Bryant and Bassik will also monitor progress and provide expertise. Further acquisition of scientific and professional skills will be achieved by utilizing the educational resources available through the Stanford University School of Medicine and Office of Postdoctoral Affairs. Stanford provides an outstanding intellectually-stimulating environment with all facilities and resources necessary for success, and all proposed training will complement Dr. Fazal?s previous knowledge in genomics and biophysics and facilitate his transition into a multidisciplinary RNA biologist in the field of spatial transcriptomics.
Many RNA species within a cell are spatially localized and locally translated into proteins, and previous studies in embryos, neurons and highly-dynamic tissues have demonstrated the vital importance of RNA subcellular location in development and cellular function. However, it has been challenging to study localization transcriptome-wide, which has hampered understanding of the extent and role of such processes in normal and diseased tissues. The proposed work will improve a newly-developed tool for the RNA community to investigate spatial transcriptomics, and use it to provide new insights into RNA biology by determining the regulatory sequences (i.e. the grammar) controlling localization, as well as identifying the associated cellular mechanisms.