Seo Jun Sung, Chua Nam-Hai
Laboratory of Plant Molecular Biology, Rockefeller University, New York, USA.
Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Singapore.
Bio Protoc. 2017 Oct 20;7(20):e2579. doi: 10.21769/BioProtoc.2579.
RNA-Protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA/protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-Protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for visualization of RNA-Protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) ( Schonberger , 2012 ). Target RNA is tagged with 6xMS2 and MCP and RNA binding protein are fused with YFP fragments. DNA constructs encoding such fusion RNA and proteins are infiltrated into tobacco leaves with suspensions. RNA-Protein interaction is observed by confocal microscope.
RNA-蛋白质相互作用在各种真核生物生物学过程中发挥着重要作用。植物中RNA/蛋白质复合物亚细胞定位的分子成像对于理解这些相互作用至关重要。然而，迄今为止尚未开发出在活体植物中对RNA-蛋白质相互作用进行成像的方法。最近，我们开发了一种三分子荧光互补（TriFC）系统，用于通过在烟草叶片中瞬时表达来可视化RNA-蛋白质相互作用。在该方法中，我们将传统的双分子荧光互补（BiFC）系统与MS2系统（噬菌体MS2外壳蛋白[MCP]及其结合RNA序列[MS2序列]）相结合（舍恩贝格尔，2012年）。靶RNA用6xMS2标记，MCP和RNA结合蛋白与YFP片段融合。编码此类融合RNA和蛋白质的DNA构建体与悬浮液一起渗入烟草叶片。通过共聚焦显微镜观察RNA-蛋白质相互作用。
