1. Hybridization-proximity labeling reveals spatially ordered interactions of nuclear RNA compartments
- Author
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Tek Hong Chung, Eugene V. Makeyev, and Karen Yap
- Subjects
Technology ,Proteome ,Nucleolus ,paraspeckles ,Computational biology ,Biology ,Proof of Concept Study ,Mass Spectrometry ,Transcriptome ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Compartment (development) ,RNA-Seq ,nucleolus ,Molecular Biology ,ascorbate peroxidase ,digoxigenin-binding domain ,RNA, Nuclear ,030304 developmental biology ,Cell Nucleus ,0303 health sciences ,Perinucleolar compartment ,perinucleolar compartment ,RNA-Binding Proteins ,RNA ,Paraspeckle ,Cell Biology ,Paraspeckles ,Cell Compartmentation ,Genetic Techniques ,RNA, Ribosomal ,RNA, Long Noncoding ,RNA-containing compartments ,higher-order nuclear organization ,proximity biotinylation ,030217 neurology & neurosurgery ,HeLa Cells ,Protein Binding - Abstract
Summary The ability of RNAs to form specific contacts with other macromolecules provides an important mechanism for subcellular compartmentalization. Here we describe a suite of hybridization-proximity (HyPro) labeling technologies for unbiased discovery of proteins (HyPro-MS) and transcripts (HyPro-seq) associated with RNAs of interest in genetically unperturbed cells. As a proof of principle, we show that HyPro-MS and HyPro-seq can identify both known and previously unexplored spatial neighbors of the noncoding RNAs 45S, NEAT1, and PNCTR expressed at markedly different levels. Notably, HyPro-seq uncovers an extensive repertoire of incompletely processed, adenosine-to-inosine-edited transcripts accumulating at the interface between their encoding chromosomal regions and the NEAT1-containing paraspeckle compartment. At least some of these targets require NEAT1 for their optimal expression. Overall, this study provides a versatile toolkit for dissecting RNA interactomes in diverse biomedical contexts and expands our understanding of the functional architecture of the mammalian nucleus., Graphical abstract, Highlights • HyPro labeling uncovers interactors and spatial neighbors of RNAs of interest • Protein and RNA partners are identified by mass spectrometry and deep sequencing • No genetic modifications are required, allowing wider biomedical use • Interactomes of RNA-containing nuclear bodies are mapped as a proof of principle, Yap et al. developed a spatial interactome mapping approach relying on recruitment of a compact peroxidase reagent to RNAs of interest and proximity biotinylation in fixed genetically unperturbed cells. The authors show that this technology can identify RNA compartment-specific proteomes and transcriptomes and reveal higher order contacts of transcribed genomic regions.
- Published
- 2022