Identification of RNA-protein interaction networks using PAR-CLIP.
Identification of RNA-protein interaction networks using PAR-CLIP.
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DOI：
10.1002/wrna.1103
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发表时间：
2012-03
影响因子：
7.3
通讯作者：
Tuschl, Thomas
中科院分区：
文献类型：
--
作者：
Ascano, Manuel;Hafner, Markus;Cekan, Pavol;Gerstberger, Stefanie;Tuschl, Thomas
所有mRNA分子都受到一定程度的转录后基因调控（PTGR），涉及剪接、切割和多聚腺苷酸化、编辑、转运、稳定性和翻译的序列依赖性调节。最近引入的深度测序技术使得能够开发新方法来广泛绘制RNA结合蛋白（RBP）及其RNA靶位点之间的相互作用位点。在这篇文章中，我们回顾了交联和免疫沉淀（CLIP）的方法适用于大规模的目标RNA结合位点和相应的RNA识别元件的识别。CLIP方法具有在单个实验中检测数十万个结合位点的潜力，尽管信号与噪声的分离可能具有挑战性。因此，每种CLIP方法都开发了不同的策略来区分真实目标和背景。我们专注于光活化的核糖核苷增强的CLIP，它依赖于细胞内掺入的光活化的核糖核苷类似物到新生的成绩单，并产生交联后，有利于分离的信号与噪声的特征序列的变化。跨成熟和初级mRNA转录物的结合位点的位置和分布的精确知识允许对所检查的RBP的细胞定位和调节功能的重要见解。当与其他系统范围的方法测量转录本和蛋白质丰度相结合时，跨转录组的高分辨率RBP结合位点图的生成将拓宽我们对PTGR的理解，从而导致干扰这些过程的遗传疾病的治疗性治疗的新策略。
All mRNA molecules are subject to some degree of post-transcriptional gene regulation (PTGR) involving sequence-dependent modulation of splicing, cleavage and polyadenylation, editing, transport, stability, and translation. The recent introduction of deep-sequencing technologies enabled the development of new methods for broadly mapping interaction sites between RNA-binding proteins (RBPs) and their RNA target sites. In this article, we review crosslinking and immunoprecipitation (CLIP) methods adapted for large-scale identification of target RNA-binding sites and the respective RNA recognition elements. CLIP methods have the potential to detect hundreds of thousands of binding sites in single experiments although the separation of signal from noise can be challenging. As a consequence, each CLIP method has developed different strategies to distinguish true targets from background. We focus on photoactivatable ribonucleoside-enhanced CLIP, which relies on the intracellular incorporation of photoactivatable ribonucleoside analogs into nascent transcripts, and yields characteristic sequence changes upon crosslinking that facilitate the separation of signal from noise. The precise knowledge of the position and distribution of binding sites across mature and primary mRNA transcripts allows critical insights into cellular localization and regulatory function of the examined RBP. When coupled with other systems-wide approaches measuring transcript and protein abundance, the generation of high-resolution RBP-binding site maps across the transcriptome will broaden our understanding of PTGR and thereby lead to new strategies for therapeutic treatment of genetic diseases perturbing these processes.
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影响因子：
64.5
作者：
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通讯作者：
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