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.
| Dominguez, Daniel; Freese, Peter; Alexis, Maria S et al. (2018) Sequence, Structure, and Context Preferences of Human RNA Binding Proteins. Mol Cell 70:854-867.e9 |
| Krach, Florian; Batra, Ranjan; Wheeler, Emily C et al. (2018) Transcriptome-pathology correlation identifies interplay between TDP-43 and the expression of its kinase CK1E in sporadic ALS. Acta Neuropathol 136:405-423 |
| Nussbacher, Julia K; Yeo, Gene W (2018) Systematic Discovery of RNA Binding Proteins that Regulate MicroRNA Levels. Mol Cell 69:1005-1016.e7 |
| 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 |
| Van Nostrand, Eric L; Nguyen, Thai B; Gelboin-Burkhart, Chelsea et al. (2017) Robust, Cost-Effective Profiling of RNA Binding Protein Targets with Single-end Enhanced Crosslinking and Immunoprecipitation (seCLIP). Methods Mol Biol 1648:177-200 |
| Zavolan, Mihaela; Graveley, Brenton R (2017) RNAs: dynamic and mutable. Genome Biol 18:226 |
| Li, Xiao; Zhou, Bing; Chen, Liang et al. (2017) GRID-seq reveals the global RNA-chromatin interactome. Nat Biotechnol 35:940-950 |
| Kapeli, Katannya; Martinez, Fernando J; Yeo, Gene W (2017) Genetic mutations in RNA-binding proteins and their roles in ALS. Hum Genet 136:1193-1214 |
| 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 |
| 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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