The recent development and application of methods based on the general principle of “crosslinking and proximity ligation” (crosslink-ligation) are revolutionizing RNA structure studies in living cells. However, extracting structure information from such data presents unique challenges. Here, we introduce a set of computational tools for the systematic analysis of data from a wide variety of crosslink-ligation methods, specifically focusing on read mapping, alignment classification, and clustering. We design a new strategy to map short reads with irregular gaps at high sensitivity and specificity. Analysis of previously published data reveals distinct properties and bias caused by the crosslinking reactions. We perform rigorous and exhaustive classification of alignments and discover eight types of arrangements that provide distinct information on RNA structures and interactions. To deconvolve the dense and intertwined gapped alignments, we develop a network/graph-based tool Crosslinked RNA Secondary Structure Analysis using Network Techniques (CRSSANT), which enables clustering of gapped alignments and discovery of new alternative and dynamic conformations. We discover that multiple crosslinking and ligation events can occur on the same RNA, generating multisegment alignments to report complex high-level RNA structures and multi-RNA interactions. We find that alignments with overlapped segments are produced from potential homodimers and develop a new method for their de novo identification. Analysis of overlapping alignments revealed potential new homodimers in cellular noncoding RNAs and RNA virus genomes in the Picornaviridae family. Together, this suite of computational tools enables rapid and efficient analysis of RNA structure and interaction data in living cells.

中文翻译：

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RNA 交联连接数据的分类和聚类揭示了复杂的结构和同源二聚体


最近基于“交联和邻近连接”（交联连接）一般原理的方法的开发和应用正在彻底改变活细胞中的 RNA 结构研究。然而，从此类数据中提取结构信息提出了独特的挑战。在这里，我们介绍了一组计算工具，用于系统分析来自各种交联连接方法的数据，特别关注读段映射、比对分类和聚类。我们设计了一种新策略，以高灵敏度和特异性绘制具有不规则间隙的短读段。对先前发布的数据的分析揭示了交联反应引起的独特特性和偏差。我们对比对进行严格而详尽的分类，并发现了八种类型的排列，它们提供了有关 RNA 结构和相互作用的独特信息。为了对密集且相互交织的间隙比对进行解卷积，我们开发了一种基于网络/图形的工具使用网络技术进行交联 RNA 二级结构分析 (CRSSANT)，该工具能够对间隙比对进行聚类并发现新的替代和动态构象。我们发现同一个 RNA 上可以发生多个交联和连接事件，生成多片段比对来报告复杂的高级 RNA 结构和多 RNA 相互作用。我们发现，重叠片段的比对是由潜在的同源二聚体产生的，并开发了一种新的方法来对其进行从头识别。对重叠比对的分析揭示了小核糖核酸病毒科的细胞非编码 RNA 和 RNA 病毒基因组中潜在的新同二聚体。 这套计算工具共同实现了对活细胞中 RNA 结构和相互作用数据的快速有效分析。