Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ [Xist OMAP ChIP]
Ontology highlight
ABSTRACT: 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.
Project description: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.
Project description: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.
Project description: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.
Project description:Within all cells, RNA molecules form complex networks of molecular interactions that are central to their function, but discovering these interactions remains challenging. 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 DNA probes to deliver proximity-biotinylating enzymes to a target RNA, enabling molecules within that RNA's subcellular microcompartment to be enriched by streptavidin pulldown. O-MAP induces exceptionally precise in situ biotinylation, and unlike alternative methods it enables straightforward optimization of its RNA-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 discovery of nucleolar-chromatin interactions across multiple cell lines. O-MAP uses exclusively off-the-shelf parts requiring no genetic- or cell-line engineering and is easily portable across diverse specimen-types and target RNAs. We therefore anticipate its application to a broad array of RNA phenomena.
Project description:Here, we introduce a method termed DNA O-MAP, which uses programmable peroxidase-conjugated oligonucleotide probes to biotinylate nearby proteins. We show that DNA O-MAP can be coupled with sample multiplexed quantitative proteomics and next-generation sequencing to quantify DNA-protein and DNA-DNA interactions at specific genomic loci.
Project description:Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ [Xist OMAP ChIP]
Project description:Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ [Xist OMAP RIP]
Project description:RNAs interact with networks of proteins to form complexes (RNPs) that govern many biological processes, but inter-protein networks on RNA are currently impossible to examine in a comprehensive way. We developed a live-cell RNP-MaP (RNP network analysis by mutational profiling) chemical probing strategy for mapping simultaneous binding by and cooperative interactions among multiple proteins with single RNA molecules at nucleotide resolution. RNP-MaP revealed that two structurally related, but sequence-divergent noncoding RNAs, RNase P and RMRP, share nearly identical RNP networks and, further, that protein-mediated structural communication identifies function-critical network hubs in these RNAs. RNP-MaP identified previously unknown protein interaction networks within the XIST long noncoding RNA that are conserved between mouse and human RNAs and defined silencing, compartmentalization and splicing communities of proteins whose binding sites are networked together on XIST. The XIST E region contains a dense network of protein interactions, and including PTBP1, MATR3, and TIA1 proteins , which RNP-MaP revealed to each bind the XIST E region via two distinct interaction modes.; Depletion of PTBP1 and MATR3 caused native XIST particles to disperse and disappear in a human cell line. the The highly networked XIST E region was sufficient to mediate XIST RNA-like foci formation in cells. RNP-MaP enables discovery and prioritization of in-cell protein interaction networks critical for function in long RNAs, in the absence of pre-existing knowledge about protein binding sites.
Project description:We here adapt Oligonucleotide-mediated proximity-interactome mapping (O-MAP), a novel RNA-targeted microenvironment-mapping method, to biochemically characterize the molecular neighborhoods near individual genomic loci. By targeting O-MAP to introns within the TTN pre-mRNA, we have systematically mapped the chromatin loci, RNAs, and proteins within a cardiomyocyte-specific "RNA factory" nucleated on these TTN-derived noncoding transcripts. This revealed an unanticipated compartmental architecture that organizes trans-chromosomal domains into a hub of transcriptionally silenced chromatin, and which recruits dozens of unique RNA-binding and chromatin-scaffolding factors, including QKI and SAFB, along with their target transcripts. Loss of the cardiac-specific splicing factor RBM20, a master regulator of TTN splicing, remodels nearly every facet of factor architecture, suggesting new mechanisms in RBM20-driven dilated cardiomyopathy. This establishes O-MAP as a pioneering method for probing single-locus, microcompartment-level interactions that are opaque to conventional tools, and suggests new mechanisms by which coding genes can "moonlight" as nuclear-architectural noncoding RNAs.
Project description:Oligonucleotide-directed proximity-interactome mapping (O-MAP): A unified method for discovering RNA-interacting proteins, transcripts and genomic loci in situ