Savulescu Anca F, Stoychev Stoyan, Mamputha Sipho, Mhlanga Musa M
Division of Chemical, Systems & Synthetic Biology, Institute for Infectious Disease & Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Biomedical Research Unit, Council for Scientific and Industrial Research, Pretoria, South Africa.
Bio Protoc. 2020 Jun 5;10(11):e3639. doi: 10.21769/BioProtoc.3639.
RNA binding proteins (RBPs) interact with cellular mRNAs, controlling various steps throughout the lifetime of these transcripts, including transcription, cellular transport, subcellular localization, translation and degradation. In addition to binding mRNA transcripts, a growing number of RBPs are shown to bind long noncoding RNAs (lncRNAs), controlling key cellular processes, including gene expression and translation of proteins. Current methodologies aimed at identifying and characterizing protein binding partners of specific RNAs of interest typically rely on tagging of the RNA with affinity aptamers, using transcribed RNA or immobilized oligonucleotides to capture RNA-protein complexes under native conditions. These assays are coupled with mass spectrometry or Western Blot analysis to identify or/and confirm interacting proteins. Here, we describe an alternative approach to identify protein binding partners of mRNAs and large long noncoding RNAs. This approach relies on biochemical pulldown of specific target RNAs and interacting protein partners from cellular lysates coupled with mass spectrometry to identify novel interacting proteins. By using 24-48 ~20 mer biotinylated DNA probes that hybridize to the target RNA, the method ensures high specificity and minimal off target binding. This approach is reproducible and fast and serves as a base for discovery studies to identify proteins that bind to RNAs of interest.
RNA结合蛋白（RBPs）与细胞中的mRNA相互作用，在这些转录本的整个生命周期中控制多个步骤，包括转录、细胞运输、亚细胞定位、翻译和降解。除了结合mRNA转录本外，越来越多的RBPs被证明可以结合长链非编码RNA（lncRNAs），从而控制关键的细胞过程，包括基因表达和蛋白质翻译。目前旨在鉴定和表征特定感兴趣RNA的蛋白质结合伴侣的方法通常依赖于用亲和适体标记RNA，使用转录的RNA或固定化的寡核苷酸在天然条件下捕获RNA-蛋白质复合物。这些分析与质谱或蛋白质印迹分析相结合，以鉴定或/和确认相互作用的蛋白质。在这里，我们描述了一种鉴定mRNA和大型长链非编码RNA的蛋白质结合伴侣的替代方法。该方法依赖于从细胞裂解物中对特定靶RNA和相互作用的蛋白质伴侣进行生化下拉，并结合质谱来鉴定新的相互作用蛋白质。通过使用与靶RNA杂交的24 - 48个约20聚体的生物素化DNA探针，该方法确保了高特异性和最小的非靶标结合。这种方法具有可重复性且快速，为发现研究提供了基础，以鉴定与感兴趣的RNA结合的蛋白质。
