Biozentrum, University of Basel and Swiss Institute of Bioinformatics, Basel, Switzerland.
Nat Methods. 2011 May 15;8(7):559-64. doi: 10.1038/nmeth.1608.
Cross-linking and immunoprecipitation (CLIP) is increasingly used to map transcriptome-wide binding sites of RNA-binding proteins. We developed a method for CLIP data analysis, and applied it to compare CLIP with photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP) and to uncover how differences in cross-linking and ribonuclease digestion affect the identified sites. We found only small differences in accuracies of these methods in identifying binding sites of HuR, which binds low-complexity sequences, and Argonaute 2, which has a complex binding specificity. We found that cross-link-induced mutations led to single-nucleotide resolution for both PAR-CLIP and CLIP. Our results confirm the expectation from original CLIP publications that RNA-binding proteins do not protect their binding sites sufficiently under the denaturing conditions used during the CLIP procedure, and we show that extensive digestion with sequence-specific RNases strongly biases the recovered binding sites. This bias can be substantially reduced by milder nuclease digestion conditions.
交联和免疫沉淀（CLIP）越来越多地用于绘制 RNA 结合蛋白的转录组范围结合位点。我们开发了一种用于 CLIP 数据分析的方法，并将其应用于比较 CLIP 与光活化核苷酸增强 CLIP（PAR-CLIP），并揭示交联和核糖核酸酶消化的差异如何影响鉴定的位点。我们发现，在鉴定 HuR（结合低复杂度序列）和 Argonaute 2（具有复杂结合特异性）的结合位点时，这些方法的准确性只有很小的差异。我们发现，交联诱导的突变导致 PAR-CLIP 和 CLIP 的单核苷酸分辨率。我们的结果证实了原始 CLIP 出版物的预期，即在 CLIP 过程中使用的变性条件下，RNA 结合蛋白不能充分保护其结合位点，并且我们表明，序列特异性核糖核酸酶的广泛消化强烈偏向于回收的结合位点。通过更温和的核酸酶消化条件可以大大降低这种偏差。
