Studying RNA-DNA interactome by Red-C identifies noncoding RNAs associated with various chromatin types and reveals transcription dynamics

Noncoding RNAs (ncRNAs) participate in various biological processes, including regulation of transcription and sustaining genome 3D organization. Here, we present a method called Red-C, which exploits proximity ligation to identify contacts with the genome for all RNA molecules present in the nucleus. Using Red-C, we uncover RNA-DNA interactome of human K562 cells and identify hundreds of ncRNAs enriched in active or repressed chromatin. We found two miRNAs, MIR3648 and MIR3867 transcribed from rRNA locus, that are associated with inactive chromatin genome-wide. These miRNAs favor bulk heterochromatin over Polycomb repressed one and interact preferentially with late-replicating genomic regions. Analysis of RNA-DNA interactome also allowed us to trace the kinetics of mRNA production. Our data support the model of co-transcriptional intron splicing, however, surprisingly, contradict the hypothesis of circularization of actively transcribed genes. Overall design: The Red-C experimental procedure for mapping 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. We applied Red-C protocol to asynchronous cell cultures (K562, normal human skin fibroblasts). The specificity of Red-C protocol was verified in control experiments with the omission of the DNA ligation step or treatment of RNA-DNA chimeras with RNase A. We also performed RNA-seq analysis of rRNA-depleted total cellular RNA from K562 cells.