RNA-protein interactions are critical for the regulation of gene expression in processes such as alternative splicing, translation regulation, RNA localization, RNA export, and RNA degradation. In order to identify the regulatory network of interactions between RNAs and proteins, I propose a rapid genome-scale method to determine the specific RNA targets and RNA binding sites of proteins. This proposal uses libraries of oligonucleotides attached to optically encoded beads. Each bead is attached to a specific oligonucleotide that can be identified by reading the optical signature in a flow cytometer. Genomic-scales will be attained by spatially arraying oligo libraries or constructing very large libraries to probe interactions in one tube. Incremental increases in library complexity will be gradually tested to reach genomic scales. A labeled genomic RNA pool will be bound to a labeled protein and then hybridized to the oligonucleotide-bead library. Beads with bound protein and/or bound RNA will be selected in a flow cytometer. The oligo sequences will be assembled to identify the bound RNA and the binding site. This approach is also amenable to detailed binding studies to explore binding on large scales. During development of the assay, biochemical experiments analyzing mRNP assembly will be pursued. In sum, the bead binding assay not only represents a novel approach to genomic screening but also a new alternative to study RNA-protein interactions.
The Specific Aims are designed to incrementally develop the bead technology while increasing our understanding of RNA-protein interactions.
Aim 2 proposes to use published microarray methods to identify RNAs bound a clinically important RNA binding protein. During the proposal period, standard genomic experiments will complement bead assay development.
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