Abstract
Non-coding RNAs (ncRNAs) participate in various biological processes, including regulating transcription and sustaining genome 3D organization. Here, we present a method termed Red-C that exploits proximity ligation to identify contacts with the genome for all RNA molecules present in the nucleus. Using Red-C, we uncovered the RNA–DNA interactome of human K562 cells and identified hundreds of ncRNAs enriched in active or repressed chromatin, including previously undescribed RNAs. Analysis of the RNA–DNA interactome also allowed us to trace the kinetics of messenger RNA production. Our data support the model of co-transcriptional intron splicing, but not the hypothesis of the circularization of actively transcribed genes.
Highlights
The vast majority of the eukaryotic genome is transcribed to produce a broad range of RNAs, including both protein-coding and non-coding RNAs (Hangauer et al 2014)
Development of Red-C The Red-C (RNA ends on DNA capture) experimental procedure for mapping the RNA– DNA interactome is based on adapter-mediated RNA–DNA ligation in fixed nuclei followed by high-throughput sequencing of the chimeric RNA–DNA molecules (Fig. 1A, Fig. S1A)
DNA-protein-RNA complexes are fixed with formaldehyde in vivo, DNA is fragmented with a restriction enzyme, and the ends are blunted and A-tailed
Summary
The vast majority of the eukaryotic genome is transcribed to produce a broad range of RNAs, including both protein-coding and non-coding RNAs (ncRNAs) (Hangauer et al 2014). Long ncRNAs (lncRNAs, > 200 nt) participate in various biological processes, from regulating enzymatic activities to sustaining genome imprinting and nuclear body biogenesis (Quinn and Chang 2016; Sun et al 2018). LncRNAs may modulate the chromatin structure by binding and targeting activator or repressor complexes to particular genomic loci (Geisler and Coller 2013; Sun et al 2018). Because they are physically linked to DNA via transcribing RNA Pol II molecules, lncRNAs may fulfill their function immediately following or during transcription without the need for processing or redistribution. Examples of cis-acting lncRNAs include lncRNAs from imprinted loci, dosage compensation lncRNAs, antisense RNAs, and autoregulatory RNAs (reviewed in (Quinn and Chang 2016))
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