This proposal seeks renewal of a multi-PI project (Fu and Yeo) to use global approaches to elucidate regulatory principles in the regulation of alternative splicing in mammalian genomes. Built upon our accomplishments in the past funding cycle, including extensive preliminary results, we propose to conduct several systematic loss- and gain-of-function studies to identify genes, gene networks, and pathways involved in the regulation of alternative splicing in three specific aims.
The first aim i s to perform large-scale network analysis of regulated alternative splicing. Using the two-dimensional mRNA isoform profiling platform developed in our labs, we propose to conduct both genome-wide RNAi and overexpression screening in HEK293 cells and score a set of commonly regulated splicing events (~400) in each treatment condition. We will also complement these genome-wide perturbation studies with transcriptomic analyses by comprehensive RNA-seq against shRNA-mediated depletion (already completed) and ectopic expression of ~300 carefully selected RNA binding proteins (RBPs). These data will help identify new splicing factors, integrate RBPs into transcription, epigenetic and signaling pathways, and decipher both unique and dominant functions of individual RBPs. In the second aim, we propose to analyze RBP-centric protein-protein interaction networks. In particular, we propose to take full advantage of our validated library of open reading frames encoding RBPs for quantitative proteomic analysis of 300 RBPs with and without RNase treatment to identify both RNA-dependent and independent interactions within the framework of RBP complexes. Using the large datasets of functional RNA targets (Aim 1) and RBP-centric protein-protein interactions (Aim 2), we will in Aim 3 perform integrated analysis of RNA genomics data to build predictive models of the regulation of alternative splicing by RBPs. We will use or develop a set of computational tools to predict novel RBP targets, refine individual RBP-central gene networks, and most importantly, integrate comprehensive RBP cis and trans interactomes with system-wide perturbation to build predictive models for cell-specific regulation of alternative splicing. We believe that such integrated analysis will have major impacts on our understanding of regulated splicing and associated disease mechanisms.

Public Health Relevance

Our RNA genomics project aims to use genomics tools to systematically elucidate genes, gene networks, and pathways involved in the regulation of alternative splicing in mammalian cells. The proposed research will provide critical molecular insights into regulated RNA processing and its coupling with other steps in gene expression, which will form the basis for development of effective treatment strategies against many RNA- related human diseases.

National Institute of Health (NIH)
National Human Genome Research Institute (NHGRI)
Research Project (R01)
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Genomics, Computational Biology and Technology Study Section (GCAT)
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Feingold, Elise A
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University of California San Diego
Other Basic Sciences
Schools of Medicine
La Jolla
United States
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Kakaradov, Boyko; Arsenio, Janilyn; Widjaja, Christella E et al. (2017) Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA sequencing. Nat Immunol 18:422-432
Song, Yan; Botvinnik, Olga B; Lovci, Michael T et al. (2017) Single-Cell Alternative Splicing Analysis with Expedition Reveals Splicing Dynamics during Neuron Differentiation. Mol Cell 67:148-161.e5
Deffit, Sarah N; Yee, Brian A; Manning, Aidan C et al. (2017) The C. elegans neural editome reveals an ADAR target mRNA required for proper chemotaxis. Elife 6:
Jiang, Li; Shao, Changwei; Wu, Qi-Jia et al. (2017) NEAT1 scaffolds RNA-binding proteins and the Microprocessor to globally enhance pri-miRNA processing. Nat Struct Mol Biol 24:816-824
Van Nostrand, Eric L; Gelboin-Burkhart, Chelsea; Wang, Ruth et al. (2017) CRISPR/Cas9-mediated integration enables TAG-eCLIP of endogenously tagged RNA binding proteins. Methods 118-119:50-59
Lardelli, Rea M; Schaffer, Ashleigh E; Eggens, Veerle R C et al. (2017) Biallelic mutations in the 3' exonuclease TOE1 cause pontocerebellar hypoplasia and uncover a role in snRNA processing. Nat Genet 49:457-464
D'Antonio, Matteo; Woodruff, Grace; Nathanson, Jason L et al. (2017) High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells. Stem Cell Reports 8:1101-1111
Van Nostrand, Eric L; Shishkin, Alexander A; Pratt, Gabriel A et al. (2017) Variation in single-nucleotide sensitivity of eCLIP derived from reverse transcription conditions. Methods 126:29-37
Bos, Tomas J; Nussbacher, Julia K; Aigner, Stefan et al. (2016) Tethered Function Assays as Tools to Elucidate the Molecular Roles of RNA-Binding Proteins. Adv Exp Med Biol 907:61-88
Sundararaman, Balaji; Zhan, Lijun; Blue, Steven M et al. (2016) Resources for the Comprehensive Discovery of Functional RNA Elements. Mol Cell 61:903-13

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