Pre-mRNA splicing is essential for proper gene expression in higher eukaryotic genomes, as the vast majority of genes contain introns that have to be accurately recognized and removed. Recent studies have revealed that >90% of the genes undergo alternative splicing, which is believed to contribute to the complexity of the proteome in different cell types and tissues in vertebrates and abundant evidence suggests that altered splicing causes a variety of human diseases. Despite extensive knowledge on the splicing mechanism based on biochemical dissection of model minigenes, we know little about how many genes are involved in the regulation of alternative splicing and where the functional RNA elements are embedded in the human genome. Built on our productive research in the current award period, we now propose a bold plan to systematically attack the critical gap of our knowledge about the regulation of alternative splicing. We will pursue three major lines of research by utilizing the latest and innovative genomics technologies. (1) We will use a new, automated platform recently developed in our lab to profile hundreds of conserved alternative splicing events against every annotated genes in the human genome. This unbiased approach will generate unprecedented information to uncover novel splicing regulators and deduce pathways in regulated splicing. (2) We will focus on RNA binding proteins involved in individual regulatory pathways to elucidate the molecular basis for regulated splicing by mapping their physical interactions with expressed RNA. For this purpose, we will construct a large panel of cell lines based on FLP-In 293 cells to express individual RNA binding proteins as a V5-tagged protein at the C- terminus, which will permit large-scale mapping of RNA-protein interactions by CLIP-seq (CrossLinking ImmunoPrecipitation followed by high throughput sequencing) under a similar and optimized set of conditions. (3) Our third goal is to use the information generated from the proposed mapping and functional studies to develop an integrated framework for de novo prediction of splicing regulation by using machine-learning and graphical models. This research has the potential to fundamentally change our view on splicing control and its contribution to human disease.

Public Health Relevance

This RNA genomics project aims to deduce the splicing code in the human genome by systematic determination of genes involved in regulated splicing, elucidation of regulatory pathways, and RNA-protein interactions. The proposed research will use the latest genomics technologies to reveal functional RNA elements in the human genome, which will provide the molecular basis for many RNA-related human diseases.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
2R01HG004659-04
Application #
8114666
Study Section
Genomics, Computational Biology and Technology Study Section (GCAT)
Program Officer
Good, Peter J
Project Start
2008-06-01
Project End
2014-05-31
Budget Start
2011-09-01
Budget End
2012-05-31
Support Year
4
Fiscal Year
2011
Total Cost
$757,461
Indirect Cost
Name
University of California San Diego
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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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