Patrucco Laura, Peano Clelia, Chiesa Andrea, Guida Filomena, Luisi Imma, Boria Ilenia, Mignone Flavio, De Bellis Gianluca, Zucchelli Silvia, Gustincich Stefano, Santoro Claudio, Sblattero Daniele, Cotella Diego
a Department of Health Sciences and Interdisciplinary Research Center on Autoimmune Diseases (IRCAD) ; Università del Piemonte Orientale ; Novara , Italy.
b Institute of Biomedical Technologies; National Research Council (ITB CNR) ; Milan , Italy.
RNA Biol. 2015;12(12):1289-300. doi: 10.1080/15476286.2015.1107702.
We describe here a platform for high-throughput protein expression and interaction analysis aimed at identifying the RNA-interacting domainome. This approach combines the selection of a phage library displaying "filtered" open reading frames with next-generation DNA sequencing. The method was validated using an RNA bait corresponding to the AU-rich element of α-prothymosin, an RNA motif that promotes mRNA stability and translation through its interaction with the RNA-binding protein ELAVL1. With this strategy, we not only confirmed known RNA-binding proteins that specifically interact with the target RNA (such as ELAVL1/HuR and RBM38) but also identified proteins not previously known to be ARE-binding (R3HDM2 and RALY). We propose this technology as a novel approach for studying the RNA-binding proteome.
我们在此描述了一个用于高通量蛋白质表达和相互作用分析的平台，旨在鉴定RNA相互作用结构域组。这种方法将展示“筛选过的”开放阅读框的噬菌体文库选择与下一代DNA测序相结合。该方法使用与α-胸腺素富含AU元件相对应的RNA诱饵进行了验证，α-胸腺素富含AU元件是一种RNA基序，通过与RNA结合蛋白ELAVL1相互作用促进mRNA稳定性和翻译。通过这种策略，我们不仅证实了与靶RNA特异性相互作用的已知RNA结合蛋白（如ELAVL1/HuR和RBM38），还鉴定出了以前未知与富含AU元件结合的蛋白（R3HDM2和RALY）。我们提出这项技术作为研究RNA结合蛋白质组的一种新方法。
