Throughout biology, RNA molecules form complex networks of molecular interactions that are central to their function, but remain challenging to investigate.
More...Throughout biology, RNA molecules form complex networks of molecular interactions that are central to their function, but remain challenging to investigate. Here, we introduce Oligonucleotide-mediated proximity-interactome MAPping (O-MAP), a straightforward method for elucidating the biomolecules near an RNA of interest, within its native cellular context. O-MAP uses programmable oligonucleotide probes to deliver proximity-biotinylating enzymes to a target RNA, enabling nearby molecules to be enriched by streptavidin pulldown. O-MAP induces exceptionally precise RNA-localized in situ biotinylation, and unlike alternative methods it enables straightforward optimization of its targeting accuracy. Using the 47S pre-ribosomal RNA and long noncoding RNA Xist as models, we develop O-MAP workflows for unbiased discovery of RNA-proximal proteins, transcripts, and genomic loci. This revealed unexpected co-compartmentalization of Xist and other chromatin-regulatory RNAs and enabled systematic characterization of nucleolar-chromatin interactions across multiple cell lines. O-MAP is portable to cultured cells, organoids, and tissues, and to RNAs of various lengths, abundances, and sequence composition. And, O-MAP requires no genetic manipulation and uses exclusively off-the-shelf parts. We therefore anticipate its application to a broad array of RNA phenomena.
Overall design: Oligonucleotide-directed proximity-interactome mapping (O-MAP) followed by streptavidin enrichment of biotinylated material and isolation of DNA or RNA for sequencing. O-MAP was performed in HeLa, 8988T, ASPC-1, Panc3.27, and SUIT2 cell lines against the internal transcribed spacer 1 of the 47S pre-ribosomal RNA. O-MAP was performed in Patski cells against the Xist transcript.
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