The Bioinformatics Centre, Department of Biology, University of Copenhagen, Copenhagen N, Denmark.
RNA. 2011 May;17(5):820-34. doi: 10.1261/rna.2387911. Epub 2011 Mar 9.
Microarray expression analyses following miRNA transfection/inhibition and, more recently, Argonaute cross-linked immunoprecipitation (CLIP)-seq assays have been used to detect miRNA target sites. CLIP and expression approaches measure differing stages of miRNA functioning-initial binding of the miRNP complex and subsequent message repression. We use nonparametric predictive models to characterize a large number of known target and flanking features, utilizing miRNA transfection, HITS-CLIP, and PAR-CLIP data. In particular, we utilize the precise spatial information provided by CLIP-seq to analyze the predictive effect of target flanking features. We observe distinct target determinants between expression-based and CLIP-based data. Target flanking features such as flanking region conservation are an important AGO-binding determinant-we hypothesize that CLIP experiments have a preference for strongly bound miRNP-target interactions involving adjacent RNA-binding proteins that increase the strength of cross-linking. In contrast, seed-related features are major determinants in expression-based studies, but less so for CLIP-seq studies, and increased miRNA concentrations typical of transfection studies contribute to this difference. While there is a good overlap between miRNA targets detected by miRNA transfection and CLIP-seq, the detection of CLIP-seq targets is largely independent of the level of subsequent mRNA degradation. Also, models built using CLIP-seq data show strong predictive power between independent CLIP-seq data sets, but are not strongly predictive for expression change. Similarly, models built from expression data are not strongly predictive for CLIP-seq data sets, supporting the finding that the determinants of miRNA binding and mRNA degradation differ. Predictive models and results are available at http://servers.binf.ku.dk/antar/.
微阵列表达分析后 miRNA 转染/抑制，以及最近 Argonaute 交联免疫沉淀（CLIP）-seq 检测已被用于检测 miRNA 靶位点。CLIP 和表达方法测量 miRNA 功能的不同阶段 - miRNP 复合物的初始结合和随后的 mRNA 抑制。我们使用非参数预测模型来描述大量已知的靶标和侧翼特征，利用 miRNA 转染、HITS-CLIP 和 PAR-CLIP 数据。特别是，我们利用 CLIP-seq 提供的精确空间信息来分析靶标侧翼特征的预测效果。我们观察到基于表达和 CLIP 的数据之间存在不同的靶标决定因素。侧翼特征，如侧翼区域保守性，是 AGO 结合的重要决定因素 - 我们假设 CLIP 实验优先选择涉及相邻 RNA 结合蛋白的强结合 miRNP-靶相互作用，这些蛋白增加交联的强度。相比之下，在基于表达的研究中，种子相关特征是主要决定因素，但在 CLIP-seq 研究中则不太重要，而转染研究中典型的 miRNA 浓度增加导致了这种差异。虽然 miRNA 转染和 CLIP-seq 检测到的 miRNA 靶标之间有很好的重叠，但 CLIP-seq 靶标的检测在很大程度上独立于随后的 mRNA 降解水平。此外，使用 CLIP-seq 数据构建的模型在独立的 CLIP-seq 数据集之间显示出很强的预测能力，但对表达变化的预测能力不强。同样，基于表达数据构建的模型对 CLIP-seq 数据集的预测能力也不强，这支持了 miRNA 结合和 mRNA 降解的决定因素不同的发现。预测模型和结果可在 http://servers.binf.ku.dk/antar/ 上获得。
