comrades-OO: An Object-Oriented R Package for Comprehensive Analysis of RNA Structure Generated using RNA crosslinking experiments

RNA molecules have secondary and tertiary structures in vivo which play a crucial role in various cellular processes such as the regulation of gene expression, RNA processing, and localization. Investigating these structures will enhance our understanding of their functions and contribute to the diagnosis and treatment of diseases caused by RNA dysregulation. However, there are no mature pipelines or packages for processing and analysing complex in vivo RNA structural data. Here, we present COMRADES Object-Oriented (comrades-OO), a novel software package for the comprehensive analysis of data derived from the COMRADES experiment. comrades-OO offers a comprehensive pipeline from raw sequencing reads to the identification of RNA structural features. It includes read processing and alignment, clustering of duplexes, data exploration, folding and comparisons of RNA structures. COMRADES-OO also enables comparisons between conditions, the identification of inter-RNA interactions, and the incorporation of reactivity data to improve structure prediction. Overall design: We recently developed Crosslinking Of Matched RNAs And Deep Sequencing (COMRADES) for in-depth RNA conformation capture in living cells (Ziv et al., 2018). COMRADES can detect base-paired regions in RNA inside the cell, using a clickable psoralen derivative to specifically crosslink double-stranded RNA, and high throughput sequencing to retrieve the base-pairing information Following in vivo crosslinking, the viral RNA is selectively captured, fragmented and subjected to a click-chemistry reaction to add a biotin tag to crosslinked regions. Crosslinked RNA duplexes are then selectively captured using streptavidin affinity purification. Half of the resulting RNA is proximity ligated, following reversal of the crosslink to create chimeric RNA templates for high throughput sequencing. The other half is used as a control, in which reversal of the crosslink precedes the proximity ligation, and accurately represents the background level of non-specific ligation.COMRADES can therefore measure (i) the structural diversity of alternative RNA conformations that coexist inside cells; (ii) short-distance, as well as long-distance (over tens of thousands of nucleotides) base-pairing within the same RNA molecule; and (iii) base-pairing between different RNA molecules, such as those of host and viral origin (Kudla et al., 2020; Ziv et al., 2018).

RNA分子在活体内存在二级与三级结构，此类结构在基因表达调控、RNA加工与定位等诸多细胞过程中发挥关键作用。对这些结构开展研究，有助于深化我们对其功能的认知，并为RNA失调相关疾病的诊断与治疗提供支撑。然而，目前尚无成熟的分析流程或工具包，可用于处理与分析复杂的活体内RNA结构数据。本研究提出面向对象的COMRADES（comrades-OO），一款全新的软件包，用于全面分析源自COMRADES实验的数据。comrades-OO提供从原始测序读段（raw sequencing reads）到RNA结构特征识别的完整分析流程，涵盖读段处理与比对、双链簇聚类、数据探索、RNA结构折叠与比对。此外，COMRADES-OO还支持不同实验条件间的比对、RNA分子间相互作用的识别，以及整合反应性数据以优化结构预测。整体实验设计：本团队近期开发了活细胞内深度RNA构象捕获技术——匹配RNA交联与深度测序（Crosslinking Of Matched RNAs And Deep Sequencing, COMRADES）（Ziv等，2018）。该技术利用可点击式补骨脂素衍生物（clickable psoralen derivative）特异性交联双链RNA（double-stranded RNA），并结合高通量测序（high throughput sequencing）技术，以获取细胞内RNA的碱基配对信息。在活体内交联完成后，研究人员会选择性捕获病毒RNA并进行片段化，随后通过点击化学反应（click-chemistry reaction）为交联区域添加生物素标签（biotin tag）。之后利用链霉亲和素亲和纯化（streptavidin affinity purification），选择性富集交联的RNA双链体。将得到的RNA分为两组：一半样本经交联逆转后进行近端连接（proximity ligation），以生成用于高通量测序的嵌合RNA模板（chimeric RNA templates）；另一半作为对照样本，先进行交联逆转再进行近端连接，以此准确反映非特异性连接的背景水平。因此，COMRADES可实现三类检测与分析：（i）细胞内共存的多种不同RNA构象的结构多样性；（ii）同一RNA分子内的短程碱基配对，以及跨越数万个核苷酸的长程碱基配对；（iii）不同RNA分子间的碱基配对，例如宿主与病毒来源的RNA分子之间的相互作用（Kudla等，2020；Ziv等，2018）。