Project Summary/Abstract The importance of RNA in regulating cellular processes and gene expression has generated tremendous interest in methods to reliably characterize RNAs and their interactions within cells. Currently available solutions utilize expensive synthetic probes and suffer from disrupted RNA structure and poor efficiency, or they require test-tube RNA synthesis and lack the structure and natural assembly that occurs within live cells. RNA-modifying enzymes provide a remarkable opportunity to be harnessed as powerful tools to covalently and selectively modify RNA with affinity probes in complex media. The RNA-TAG (Transglycosylation At Guanosine) methodology, recently developed in our lab, is unique in its use of bacterial tRNA Guanine Transglycosylase (TGT) to directly and selectively transfer a small covalent modification, such as a biotin, to RNA bearing a specific encoded recognition element. TGT shows promise due to the ease of derivatization of its known substrate, preQ1, as well as its substrate selection, which is orthogonal to eukaryotic systems. Building on our initial work, we seek to establish an RNA-centric technology that can facilitate the robust purification and proteomic analysis of RNA-protein complexes by direct labeling of a minimally perturbing recognition element encoded within an RNA of interest. Using CRISPR-Cas9, we will encode the TGT recognition element into the gene for the oncogenic long noncoding RNA (lncRNA) HOTAIR to facilitate labeling and isolation of the natively expressed lncRNA in complex with its bound proteins. Preliminary experiments using the novel RNA-TAG methodology have established the ability to efficiently biotinylate and pull down expressed mRNA from cellular lysate. Furthermore, we have demonstrated covalent labeling of a minimally modified HOTAIR mutant without the need for introduction of exogenous structural elements. Through the proposed work, we will establish RNA-TAG as a robust RNA labeling methodology to foster future studies in RNA biology and provide a more comprehensive understanding of how RNA contributes to the complexity of biology and human health.