Gladstone Institute of Virology and Immunology, Gladstone Institutes, San Francisco, CA, USA.
Department of Microbiology and Plant Pathology, University of California, Riverside, CA, USA.
Methods Mol Biol. 2021;2170:213-218. doi: 10.1007/978-1-0716-0743-5_15.
Protein-protein interactions (PPI) are vital in regulating the biological and physiological functions in a given cell or organism. Proteomics, in conjunction with bioinformatic tools, represents the study involving the characterization of the protein content of the genome of a given biological system. Like PPI, an interaction between either coding or noncoding RNA and a complex set of host proteins protein plays an essential role in gene expression at translational, posttranscriptional, and epigenetic level. Although a wide range of techniques such as shotgun proteomics, MuDPIT, etc. are available for characterizing PII, those for characterizing RNA-protein interactions are infancy. Given the significance of the long noncoding RNAs (lnc-RNA) in plant biology, it is imperative to isolate and characterize the functionality of the host proteome interacting with RNA. In this context, riboproteomics approach becomes a valuable tool to study these interactions. Here, using a noncoding plant pathogenic satellite-RNA (Sat-RNA) of Cucumber mosaic virus (CMV) as an RNA source, we describe a stepwise protocol for identifying the host proteome interacting specifically with the Sat-RNA. This protocol streamlines steps starting from in vitro transcription of RNA, preparation of RNA affinity column, preparation of cell lysate from Nicotiana benthamiana leaves infected with the Sat-RNA followed by the Co-IP and preparation of samples for LC-MS/MS. We believe this approach is applicable to a wide range of RNAs of any nature associated with eukaryotic and prokaryotic organisms.
蛋白质-蛋白质相互作用（PPI）对于调节特定细胞或生物体的生物和生理功能至关重要。蛋白质组学与生物信息学工具相结合，代表了对特定生物系统基因组中蛋白质含量进行特征描述的研究。与 PPI 类似，编码或非编码 RNA 与复杂的一组宿主蛋白之间的相互作用在翻译、转录后和表观遗传水平上对基因表达起着至关重要的作用。尽管有广泛的技术，如鸟枪法蛋白质组学、MuDPIT 等，可用于表征 PPI，但用于表征 RNA-蛋白质相互作用的技术仍处于起步阶段。鉴于长非编码 RNA（lnc-RNA）在植物生物学中的重要性，分离和表征与 RNA 相互作用的宿主蛋白质组是当务之急。在这种情况下，核糖体蛋白质组学方法成为研究这些相互作用的有价值的工具。在这里，我们使用黄瓜花叶病毒（CMV）的非编码植物病原卫星-RNA（Sat-RNA）作为 RNA 来源，描述了一种逐步方案，用于鉴定与 Sat-RNA 特异性相互作用的宿主蛋白质组。该方案简化了从 RNA 体外转录、RNA 亲和柱制备、受 Sat-RNA 感染的烟草叶片细胞裂解物制备开始的步骤，随后进行 Co-IP 和 LC-MS/MS 样品制备。我们相信这种方法适用于与真核和原核生物相关的任何性质的广泛的 RNA。
