Protein-RNA networks revealed through covalent RNA marks.
Protein-RNA networks revealed through covalent RNA marks.
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DOI：
10.1038/nmeth.3651
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发表时间：
2015-12
期刊：
影响因子：
48
通讯作者：
Wickens M
中科院分区：
文献类型：
--
作者：
Lapointe CP;Wilinski D;Saunders HA;Wickens M
蛋白质-RNA网络在生物防治中无处不在，处于核心地位。我们提出了一种称为“RNA标签”的方法，它通过分析纯化的细胞RNA来识别体内蛋白质-RNA的相互作用，而不需要蛋白质纯化或交联。一种感兴趣的RNA结合蛋白被融合到一种酶上，该酶将尿氨酸添加到RNA的末端。体内嵌合蛋白结合的RNA靶标用尿苷共价标记，然后使用高通量测序从提取的RNA中鉴定。我们使用这种方法鉴定了数百个与酿酒酵母PUF蛋白Puf3p结合的RNA。该方法表明，虽然RNA结合蛋白能有效地结合特定的RNA来控制其功能，但它们也对RNA进行“采样”，而不起到调节作用。我们利用这种方法发现了数百个新的和可能受调控的蛋白质靶标，这些靶标没有典型的RNA结合域Bfr1p。RNA标签方法非常适合于体内蛋白质-RNA网络的检测和分析。
Protein-RNA networks are ubiquitous and central in biological control. We present an approach, termed “RNA Tagging,” that identifies protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or crosslinking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA using high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The method revealed that while RNA-binding proteins productively bind specific RNAs to control their function, they also “sample” RNAs without exerting a regulatory effect. We exploited the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. The RNA Tagging approach is well-suited to detect and analyze protein-RNA networks in vivo.