A computational approach for identifying microRNA-target interactions using high-throughput CLIP and PAR-CLIP sequencing.
A computational approach for identifying microRNA-target interactions using high-throughput CLIP and PAR-CLIP sequencing.
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DOI：
10.1186/1471-2164-14-s1-s2
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发表时间：
2013
期刊：
影响因子：
4.4
通讯作者：
Huang HD
中科院分区：
文献类型：
--
作者：
Chou CH;Lin FM;Chou MT;Hsu SD;Chang TH;Weng SL;Shrestha S;Hsiao CC;Hung JH;Huang HD
microRNA（miRNAs）在下调基因表达中起着关键作用。通过与Argonaute家族蛋白偶联，miRNA结合到mRNA上的靶位点并进行翻译抑制。通过交联免疫沉淀法（CLIP）和光活化核糖核苷增强的CLIP法（PAR-CLIP）沿着下一代测序法（NGS）已经鉴定出大量的miRNA-靶相互作用（MTIs）。PAR-CLIP显示出高效率的RNA免疫共沉淀，但它也导致在miRNA-RNA-蛋白质交联区域中的T至C转化。这种人为错误明显降低了读段的可映射性。然而，仍然需要一种特定的工具来分析CLIP和PAR-CLIP数据，该工具将T到C的转换考虑在内。我们在此提出了第一个专门用于miRNA靶标分析的CLIP和PAR-CLIP测序分析平台，即miRTarCLIP。从头开始，它自动从原始读段中删除衔接子序列，过滤低质量读段，将C还原为T，将读段与3 'UTR对齐，扫描读段簇，识别高置信度的miRNA靶位点，并提供来自外部数据库的注释。通过多线程技术和我们新颖的C到T反转过程，miRTarCLIP与传统方法相比大大缩短了运行时间。此外，miRTarCLIP还提供基于Web的界面，以提供更好的用户浏览和搜索感兴趣的miRNA靶点的体验。为了证明miRTarCLIP的上级功能，我们将miRTarCLIP应用于两个公开可用的CLIP和PAR-CLIP测序数据集。miRTarCLIP不仅以更快的速度显示出与其他现有工具相当的结果，而且还揭示了这些推定的靶位点中的有趣特征。具体地，我们使用miRTarCLIP来揭示miRNA靶位点的位置1-7内和位置8-14内的T到C转换是显著不同的（p值= 0.02），并且当仅关注前102个高表达的miRNA靶向的位点时甚至更显著（p值= 0.01）。这些结果符合先前的发现，并进一步表明，结合miRNA表达和PAR-CLIP数据可以提高miRNA靶点预测的准确性。综上所述，我们设计了一种从CLIP-seq和PAR-CLIP测序数据中挖掘miRNA靶位点的系统方法，并将工作流程与基于Web的图形浏览器集成在一起，该浏览器提供了用户友好的界面和详细的MTI注释。我们还通过现实生活中的例子表明，miRTarCLIP是理解miRNA的强大工具。我们的集成工具可以在http://miRTarCLIP.mbc.nctu.edu.tw上免费在线访问。
MicroRNAs (miRNAs) play a critical role in down-regulating gene expression. By coupling with Argonaute family proteins, miRNAs bind to target sites on mRNAs and employ translational repression. A large amount of miRNA-target interactions (MTIs) have been identified by the crosslinking and immunoprecipitation (CLIP) and the photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) along with the next-generation sequencing (NGS). PAR-CLIP shows high efficiency of RNA co-immunoprecipitation, but it also lead to T to C conversion in miRNA-RNA-protein crosslinking regions. This artificial error obviously reduces the mappability of reads. However, a specific tool to analyze CLIP and PAR-CLIP data that takes T to C conversion into account is still in need. We herein propose the first CLIP and PAR-CLIP sequencing analysis platform specifically for miRNA target analysis, namely miRTarCLIP. From scratch, it automatically removes adaptor sequences from raw reads, filters low quality reads, reverts C to T, aligns reads to 3'UTRs, scans for read clusters, identifies high confidence miRNA target sites, and provides annotations from external databases. With multi-threading techniques and our novel C to T reversion procedure, miRTarCLIP greatly reduces the running time comparing to conventional approaches. In addition, miRTarCLIP serves with a web-based interface to provide better user experiences in browsing and searching targets of interested miRNAs. To demonstrate the superior functionality of miRTarCLIP, we applied miRTarCLIP to two public available CLIP and PAR-CLIP sequencing datasets. miRTarCLIP not only shows comparable results to that of other existing tools in a much faster speed, but also reveals interesting features among these putative target sites. Specifically, we used miRTarCLIP to disclose that T to C conversion within position 1-7 and that within position 8-14 of miRNA target sites are significantly different (p value = 0.02), and even more significant when focusing on sites targeted by top 102 highly expressed miRNAs only (p value = 0.01). These results comply with previous findings and further suggest that combining miRNA expression and PAR-CLIP data can improve accuracy of the miRNA target prediction. To sum up, we devised a systematic approach for mining miRNA-target sites from CLIP-seq and PAR-CLIP sequencing data, and integrated the workflow with a graphical web-based browser, which provides a user friendly interface and detailed annotations of MTIs. We also showed through real-life examples that miRTarCLIP is a powerful tool for understanding miRNAs. Our integrated tool can be accessed online freely at http://miRTarCLIP.mbc.nctu.edu.tw.