Vourekas Anastassios, Mourelatos Zissimos
Department of Pathology and Laboratory Medicine, Division of Neuropathology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Methods Mol Biol. 2014;1093:73-95. doi: 10.1007/978-1-62703-694-8_7.
Piwi proteins, such as Aubergine in Drosophila and Miwi and Mili in mice, form a major subclade of the Argonaute family, which comprise a distinct class of RNA-binding proteins (RBPs) able to bind small RNAs. Small RNAs can target complementary RNAs. Piwis are essential for the animal germline and bind Piwi-interacting RNAs (piRNAs) to form pi-RiboNucleoProteins (piRNPs). Although many piRNAs target retrotransposons for safeguarding genome integrity of the germ cell, whether piRNAs can target other mRNAs for regulatory purposes is still under investigation. Here we present the technical protocol for "High Throughput Sequencing after in vivo Crosslinking and Immunoprecipitation" (HITS-CLIP, CLIP-Seq), adapted for mouse Piwi proteins Mili and Miwi. We also provide general recommendations for the application of this protocol for different RBPs and also for the bioinformatic analysis of the deep sequencing data.
Piwi蛋白，如果蝇中的茄子蛋白以及小鼠中的Miwi和Mili蛋白，构成了AGO蛋白家族的一个主要亚类，该家族包含一类独特的能够结合小RNA的RNA结合蛋白（RBP）。小RNA可以靶向互补RNA。Piwi蛋白对于动物生殖系至关重要，它与Piwi相互作用RNA（piRNA）结合形成pi核糖核蛋白（piRNP）。尽管许多piRNA靶向逆转录转座子以保护生殖细胞的基因组完整性，但piRNA是否能靶向其他mRNA用于调控目的仍在研究中。在此，我们展示了适用于小鼠Piwi蛋白Mili和Miwi的“体内交联与免疫沉淀后高通量测序”（HITS-CLIP，CLIP-Seq）技术方案。我们还针对该方案在不同RBP中的应用以及深度测序数据的生物信息学分析提供了一般性建议。
