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. 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.

非编码RNA（Noncoding RNAs，ncRNAs）参与诸多生物学过程，包括转录调控与维持基因组三维空间构象。在此，我们介绍一种名为Red-C的实验方法，该方法利用邻近连接（proximity ligation）技术，鉴定细胞核内所有RNA分子与基因组的相互作用位点。借助Red-C技术，我们解析了人类K562细胞的RNA-DNA互作组，并鉴定出数百种富集于活化染色质或阻遏染色质的非编码RNA。我们发现两种由核糖体RNA（rRNA）基因座转录而来的微小RNA（miRNAs）：MIR3648与MIR3867，它们在全基因组范围内与非活化染色质相关联。相较于多梳蛋白（Polycomb）介导的阻遏染色质，这类微小RNA更倾向于结合大块异染色质，且优先与复制晚期的基因组区域发生相互作用。对RNA-DNA互作组的分析还使我们能够追踪mRNA生成的动力学过程。我们的数据支持共转录内含子剪接模型，但却出人意料地与活跃转录基因的环化假说相悖。用于绘制RNA-DNA互作组图谱的Red-C实验流程，基于固定细胞核内的接头介导的RNA-DNA连接技术，随后对嵌合RNA-DNA分子进行高通量测序。我们将Red-C实验方案应用于非同步化细胞培养体系，包括人类K562细胞与正常人类皮肤成纤维细胞。Red-C实验方案的特异性通过对照实验得以验证：对照实验分别省略DNA连接步骤，或使用核糖核酸酶A（RNase A）处理RNA-DNA嵌合分子。我们还对K562细胞中经核糖体RNA去除处理的总细胞RNA进行了RNA测序（RNA-seq）分析。