CLLASH reveals the in vivo RNA-RNA interactions recognized by hnRNPK

RNA-RNA interactions associated with various RNA-binding proteins (RBPs) regulate gene expression and critical biological processes. However, identifying RNA interactions associate with specific proteins of interest on a transcriptomic scale still remains challenging. Here we present CLLASH, a biochemical technique for transcriptome-wide identification of intra- and intermolecular RNA-RNA interactions mediated by RBPs. Applying this method in human cells reveals a remarkable landscape of intermolecular RNA interactions recognized by hnRNPK, involving all major classes of ncRNA and mRNA. We validated one new lncRNA-mRNA interaction using single-molecule RNA FISH and miRNA target genes by inhibiting this miRNA and detecting the transcription level of target genes. Our study introduces CLLASH as a widely applicable method for discovering new RNA interactions bound by defined proteins of interest.

与各类RNA结合蛋白（RNA-binding protein, RBP）相关的RNA-RNA相互作用，可调控基因表达与关键生命过程。然而，在转录组规模下鉴定与特定目标蛋白相关的RNA相互作用，仍是一项极具挑战性的工作。本研究介绍了CLLASH技术——一种可在全转录组范围内鉴定由RBPs介导的分子内与分子间RNA-RNA相互作用的生化实验方法。将该方法应用于人类细胞后，研究揭示了由异质性细胞核核糖蛋白K（heterogeneous nuclear ribonucleoprotein K, hnRNPK）识别的分子间RNA相互作用的独特图谱，该图谱涵盖了所有主要类别的非编码RNA（non-coding RNA, ncRNA）与信使RNA（messenger RNA, mRNA）。我们利用单分子RNA荧光原位杂交（single-molecule RNA fluorescence in situ hybridization, RNA FISH）验证了一种全新的长链非编码RNA（long non-coding RNA, lncRNA）与信使RNA之间的相互作用；同时通过抑制该微小RNA（microRNA, miRNA）并检测靶基因的转录水平，验证了其作为该miRNA靶基因的相关相互作用。本研究证实，CLLASH技术可作为一种普适性方法，用于发现由特定目标蛋白结合的全新RNA相互作用。