This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are extending our prior work on genomic DNA footprinting to study RNA structure and RNA-protein interactions. Because individual transcription factor binding sites are readily identified by genomic DNA footprinting, analogous methods for in vivo RNA footprinting may reveal RNA protection patterns caused by protein binding. In these experiments, cellular extracts containing RNA-protein complexes will be treated with RNaseI, and released fragments will be isolated and sequenced to identify cleavage sites. Similar to genomic DNA footprinting, sites of putative RNA-protein interaction will then be identified as short stretches of sequence (e.g. 8-20 nt) protected from cleavage. The primary goal will be to generate a detailed map of RNA-protein interaction, followed by the identification of features common to subsets of footprinted RNAs. For example, mRNAs known to be trafficked to specific sub-cellular regions may share similar footprint patterns through the binding of common regulatory proteins. To determine which proteins are bound to specific sites, we will use the recently CLIP-SEQ assay to identify candidate RNA binding proteins. This general protocol should provide an unprecedented view of RNA-protein interaction topology, while simultaneously narrowing the sites of interaction to single base-pair resolution.
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