ABSTRACT Cells are exquisitely-organized entities, with the location of biomolecules within intimately tied to the maintenance of the organism and the regulation of its genes. Where an RNA is located within the cell determines whether it will be stored, processed, spliced, or degraded. Although early studies in the 80s and 90s established the cis- and trans- regulators associated with localization of specific RNAs, for the vast majority of mRNAs, we do not know how they get to their destinations and what confers specificity and selectivity in space and time. Such location-based RNA regulation continues to pose new mysteries for the field yet promises to reveal insights into fundamental cell biology and disease mechanisms. Consequently, during his postdoc, Dr. Furqan Fazal developed an RNA-seq-based technology exploiting APEX (APEX-seq), a peroxidase enzyme, to spatially tag RNAs and generate subcellular transcriptomes in living human cells at nm-spatial and minute-temporal resolution (Fazal et al., Cell 2019). A preliminary atlas of localization revealed that thousands of genes and transcript isoforms show differential RNA subcellular localization, which has profound consequences for our understanding of development, neurodegeneration, and cancers. This proposal outlines a five-year research program for Dr. Fazal, an early-stage investigator, to determine the role of cytoskeletal motor proteins in RNA subcellular localization. Investigating molecular motors is likely to yield significant findings in the field since they have been shown to transport mRNAs in singletons both in vitro reconstituted systems and inside cells. These motors include kinesins and cytoplasmic dynein that walk on microtubules and processive myosins that traverse actin filaments. However, it is unclear inside cells what the mRNA selectively pattern of these motors is, whether they cooperate or compete, and what cofactors confer specificity of location. In this proposal, we will investigate the role of motor proteins in RNA localization. As a model system, we will focus on mRNA transport to the mitochondria, an essential organelle that has over 1000 species of mRNAs associated with its outer mitochondrial membrane (OMM). Program IA will focus on identifying the RNA cargo and individual contributions of the motors. Program IB will use inducible degradable versions of motor proteins and their associated cargos to identify the exact mechanisms of RNA transport by motors, whether by active transport, organelle hitchhiking, translation, or others. Program II will study the protein and RNA interactome of motor proteins to identify sequence (cis-) elements and protein (trans-) factors important for RNA localization. Using a combination of RNA proximity labeling using APEX-seq along with computational, chemical, genetic, and biochemical experiments, we expect to decipher the scope and regulation of RNA transport by molecular motors. As mutations in motors and their associated interactors and RNA-binding proteins (RBPs) have been linked to several diseases associated with mRNA transport and localization, our experiments will inform how such mutations disrupt these processes in disease.

PROJECT NARRATIVE 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 apply 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 contribution and regulation of cytoskeletal motor proteins and associated factors to actively transport specific RNAs to their destinations.