Background A critical step in uncovering rules of RNA processing is to study the in vivo regulatory networks of RNA binding proteins (RBPs). Crosslinking and immunoprecipitation (CLIP) methods enable mapping RBP targets transcriptome-wide, but methodological differences present challenges to large-scale analysis across datasets. The development of enhanced CLIP (eCLIP) enabled the mapping of targets for 150 RBPs in K562 and HepG2, creating a unique resource of RBP interactomes profiled with a standardized methodology in the same cell types. Results Our analysis of 223 eCLIP datasets reveals a range of binding modalities, including highly resolved positioning around splicing signals and mRNA untranslated regions that associate with distinct RBP functions. Quantification of enrichment for repetitive and abundant multicopy elements reveals 70% of RBPs have enrichment for non-mRNA element classes, enables identification of novel ribosomal RNA processing factors and sites, and suggests that association with retrotransposable elements reflects multiple RBP mechanisms of action. Analysis of spliceosomal RBPs indicates that eCLIP resolves AQR association after intronic lariat formation, enabling identification of branch points with single-nucleotide resolution, and provides genome-wide validation for a branch point-based scanning model for 3′ splice site recognition. Finally, we show that eCLIP peak co-occurrences across RBPs enable the discovery of novel co-interacting RBPs. Conclusions This work reveals novel insights into RNA biology by integrated analysis of eCLIP profiling of 150 RBPs with distinct functions. Further, our quantification of both mRNA and other element association will enable further research to identify novel roles of RBPs in regulating RNA processing.

中文翻译：

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通过分析 150 个 RNA 结合蛋白的增强 CLIP 图谱得出 RNA 处理原理


背景 揭示 RNA 加工规则的关键一步是研究 RNA 结合蛋白 (RBP) 的体内调控网络。交联和免疫沉淀 (CLIP) 方法能够在转录组范围内绘制 RBP 靶标，但方法上的差异给跨数据集的大规模分析带来了挑战。增强型 CLIP (eCLIP) 的开发实现了 K562 和 HepG2 中 150 个 RBP 靶标的映射，从而创建了在相同细胞类型中使用标准化方法进行分析的独特 RBP 相互作用组资源。结果我们对 223 个 eCLIP 数据集的分析揭示了一系列结合方式，包括剪接信号周围的高分辨率定位和与不同 RBP 功能相关的 mRNA 非翻译区域。对重复和丰富的多拷贝元件富集的量化揭示了 70% 的 RBP 对非 mRNA 元件类别进行了富集，能够识别新的核糖体 RNA 加工因子和位点，并表明与逆转录转座元件的关联反映了多种 RBP 作用机制。剪接体 RBP 分析表明，eCLIP 在内含子套索形成后解析了 AQR 关联，从而能够以单核苷酸分辨率识别分支点，并为基于分支点的扫描模型提供全基因组验证，以进行 3' 剪接位点识别。最后，我们表明，eCLIP 跨 RBP 的峰值共现使得能够发现新的共同相互作用的 RBP。结论 这项工作通过对 150 个具有不同功能的 RBP 进行 eCLIP 分析的综合分析，揭示了对 RNA 生物学的新见解。此外，我们对 mRNA 和其他元件关联的量化将使进一步的研究能够确定 RBP 在调节 RNA 加工中的新作用。