The goal of this proposal is to comprehensively characterize the functional sequence elements encoded in the human genome that are recognized by 250 RNA binding proteins (RBPs) in two cell lines. To do this, we will generate stable HeLa-S3 and GM 12878 cell lines expressing epitope-tagged RBPs and determine the sub cellular localization pattern of each RBP. These cells will be used to perform CLIP-Seq assays to define genome-wide, and at single-nucleotide resolution, the RNA sequence elements recognized by 250 RBPs. The RNA sequence elements identified will be validated using sequence-based in vitro binding assays. Furthermore, ChlP-Seq will be performed for all nuclear localized RBPs to determine the regions of the genome and chromatin that each RBP associates with. These binding assays will be supplemented with functional assays in RBP-depleted cells that will be critical for assigning functions to the identified binding sites. These assays include RNA-Seq of total cellular RNA and RNA purified from various cellular fractions, ribosomal footprint profiling, and Gro-Seq. Together, these assays will provide functional information regarding the roles of each RBP in splicing, cleavage and polyadenylation, RNA stability, RNA editing, translation, RNA localization, and transcription. Bioinformatic analysis will be performed, largely using software generated by our group, to quantitate all assays and to associate functions to the sequence elements identified in the binding assays. Together, these experiments will provide a comprehensive and in depth measure of the functions of approximately half of the human RBPs and the functional sequence elements that they interact with. This project will fill a major gap in the catalog of functional elements encoded in the human genome that are being characterized by the ENCODE consortium. The product of this project will be a unique and valuable community resource that will push the field forward in new and exciting ways and will almost certainly create new paradigms regarding the functions of RBPs and RNA-protein networks in human biology and disease.
Many human diseases are caused by defects in RNA biology. However, our understanding of what causes such diseases is limited because we lack a fundamental knowledge of the function of most RNA binding proteins. Our comprehensive survey of the biology of 250 RNA binding proteins will provide significant new insight into RNA biology and increased understanding of the causes of many human diseases.
|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|
|Graveley, Brenton R (2016) RNA Matchmaking: Finding Cellular Pairing Partners. Mol Cell 63:186-9|
|Conway, Anne E; Van Nostrand, Eric L; Pratt, Gabriel A et al. (2016) Enhanced CLIP Uncovers IMP Protein-RNA Targets in Human Pluripotent Stem Cells Important for Cell Adhesion and Survival. Cell Rep 15:666-79|
|Kapeli, Katannya; Pratt, Gabriel A; Vu, Anthony Q et al. (2016) Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses. Nat Commun 7:12143|
|Taliaferro, J Matthew; Vidaki, Marina; Oliveira, Ruan et al. (2016) Distal Alternative Last Exons Localize mRNAs to Neural Projections. Mol Cell 61:821-33|
|Teng, Mingxiang; Love, Michael I; Davis, Carrie A et al. (2016) A benchmark for RNA-seq quantification pipelines. Genome Biol 17:74|
|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|
Showing the most recent 10 out of 26 publications