Laboratory of RNA Molecular Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA.
Wiley Interdiscip Rev RNA. 2012 Mar-Apr;3(2):159-77. doi: 10.1002/wrna.1103. Epub 2011 Dec 27.
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.
所有的 mRNA 分子都受到一定程度的转录后基因调控（PTGR），包括依赖于序列的剪接、切割和多聚腺苷酸化、编辑、运输、稳定性和翻译的调节。最近引入的深度测序技术使开发新方法来广泛映射 RNA 结合蛋白（RBPs）与其 RNA 靶位点之间的相互作用位点成为可能。在本文中，我们回顾了适应于大规模鉴定靶 RNA 结合位点和相应 RNA 识别元件的交联和免疫沉淀（CLIP）方法。CLIP 方法有可能在单个实验中检测到数十万的结合位点，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的靶标和背景。我们专注于光激活核苷酸增强的 CLIP，它依赖于细胞内将光激活核苷酸类似物掺入新生转录本中，并且在交联时产生特征序列变化，有助于信号与噪声的分离。结合其他测量转录本和蛋白质丰度的全系统方法，在整个转录组中生成高分辨率 RBP 结合位点图谱，将拓宽我们对 PTGR 的理解，并为治疗这些过程中受干扰的遗传疾病的新治疗策略提供依据。
