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.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
2R01HG004659-07
Application #
8773860
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
Feingold, Elise A
Project Start
2008-06-01
Project End
2017-06-30
Budget Start
2014-09-01
Budget End
2015-06-30
Support Year
7
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Zhang, Zhen-Ning; Freitas, Beatriz C; Qian, Hao et al. (2016) Layered hydrogels accelerate iPSC-derived neuronal maturation and reveal migration defects caused by MeCP2 dysfunction. Proc Natl Acad Sci U S A 113:3185-90
Bardy, C; van den Hurk, M; Kakaradov, B et al. (2016) Predicting the functional states of human iPSC-derived neurons with single-cell RNA-seq and electrophysiology. Mol Psychiatry 21:1573-1588
Van Nostrand, Eric L; Pratt, Gabriel A; Shishkin, Alexander A et al. (2016) Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13:508-14
Dickey, Audrey S; Pineda, Victor V; Tsunemi, Taiji et al. (2016) PPAR-δ is repressed in Huntington's disease, is required for normal neuronal function and can be targeted therapeutically. Nat Med 22:37-45
Xue, Yuanchao; Qian, Hao; Hu, Jing et al. (2016) Sequential regulatory loops as key gatekeepers for neuronal reprogramming in human cells. Nat Neurosci 19:807-15
Sundararaman, Balaji; Zhan, Lijun; Blue, Steven M et al. (2016) Resources for the Comprehensive Discovery of Functional RNA Elements. Mol Cell 61:903-13
Batra, Ranjan; Stark, Thomas J; Clark, Elizabeth et al. (2016) RNA-binding protein CPEB1 remodels host and viral RNA landscapes. Nat Struct Mol Biol 23:1101-1110
Martinez, Fernando J; Pratt, Gabriel A; Van Nostrand, Eric L et al. (2016) Protein-RNA Networks Regulated by Normal and ALS-Associated Mutant HNRNPA2B1 in the Nervous System. Neuron 92:780-795
Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C et al. (2016) SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes. Mol Cell 64:282-293
Broughton, James P; Lovci, Michael T; Huang, Jessica L et al. (2016) Pairing beyond the Seed Supports MicroRNA Targeting Specificity. Mol Cell 64:320-333

Showing the most recent 10 out of 78 publications