Discovering the interactions between circRNAs and RNA-binding proteins from RNA-seq and CLIP-seq data. Homo sapiens

Although tens of thousands of circular RNAs (circRNAs) have been identified in mammalian genomes, only a few have been functionally characterized. Here, we report a new approach, circScan, to identify regulatory interactions between circRNAs and RNA-binding proteins (RBPs). Our method identifies back-splicing reads from crosslinking and immunoprecipitation followed by high-throughput sequencing (CLIP-seq) data. Using our method, we systematically scanned ~1,500 CLIP-seq datasets and identified ~12,540 and ~1,090 novel circRNA-RBP interactions in the human and mouse genomes, respectively, including all known interactions between circRNAs and Argonaute (AGO) proteins. Furthermore, more than twenty novel interactions were experimentally confirmed by RNA immunoprecipitation combined with quantitative PCR (RIP-qPCR). Importantly, we determined that natural circRNAs interact with the cap-independent translation factors eukaryotic initiation factor 3 (eIF3) and N6-methyladenosine (m6A), thus suggesting that those circRNAs may be translated into proteins. These findings demonstrate that circRNAs are regulated by various RBPs and therefore may play important roles in diverse biological processes. Overall design: Discovery of circular RNAs in 2 cell types.

尽管已在哺乳动物基因组中鉴定出数以万计的环状RNA（circular RNAs, circRNAs），但仅有极少数被解析了生物学功能。本研究报道了一种全新的分析方法——circScan，用于鉴定环状RNA与RNA结合蛋白（RNA-binding proteins, RBPs）之间的调控互作关系。该方法可从紫外交联免疫沉淀结合高通量测序（crosslinking and immunoprecipitation followed by high-throughput sequencing, CLIP-seq）数据中识别反向剪接读段。借助此方法，我们系统分析了约1500组CLIP-seq数据集，分别在人类和小鼠基因组中鉴定出约12540组和1090组全新的circRNA-RBP调控互作，其中涵盖了目前已知的所有环状RNA与Argonaute（AGO）蛋白之间的互作。此外，通过RNA免疫沉淀联合定量PCR（RNA immunoprecipitation combined with quantitative PCR, RIP-qPCR）实验验证了二十余组全新的互作关系。值得注意的是，我们发现天然环状RNA可与不依赖帽子结构的翻译因子真核起始因子3（eukaryotic initiation factor 3, eIF3）以及N6-甲基腺嘌呤（N6-methyladenosine, m6A）结合，这提示此类环状RNA可能具备编码蛋白质的能力。上述研究结果表明，环状RNA可受多种RNA结合蛋白调控，进而在多样的生物学过程中发挥关键作用。整体实验设计：在2种细胞类型中开展环状RNA的发现研究。