Mechanisms of Cell-type-specific pre-mRNA Splicing Abstract: The overarching goal of this project is to uncover cis- and trans-regulatory mechanisms of pre-mRNA splicing at the cell-type level. While single-cell RNA Sequencing (scRNA-Seq) is revolutionizing our understanding of cell- type heterogeneity in animal tissues, the extent and mechanism of cell-type-specific splicing remain largely uncharted. The major challenges are threefold: 1) Current scRNA-Seq platforms are predominantly built on read counts of the 3’ or 5’ end fragments of polyadenylated RNAs and do not have sufficient coverage for splice junctions; 2) Homologous RNA binding proteins (RBPs) frequently have overlapping expression patterns and redundant functions, making it challenging to uncover their full functions in vivo; and 3) Protein-RNA interaction has been predominantly studied in cell lines or bulk tissues using UV-crosslinking and immunoprecipitation- based approaches, and it remains a challenge to identify RBP targets in specific cell types from intact tissues. My group uses the mouse brain as a model system and has been developing new tools to overcome these challenges. We have made proof-of-concept progress and seek to 1) uncover cis-regulatory elements and coordinated splicing patterns by single-cell long-read sequencing; 2) study redundant RBP functions by multiplexed genome editing; and 3) investigate protein-RNA interaction at the cell-type level by dual RNA- deaminase editing and sequencing. Successful completion of this project will generate new tools and datasets to understand the mechanisms of cell-type-specific pre-mRNA processing. The MIRA funding mechanism will permit the flexibility to integrate technological advances and study new biological questions.