Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA.
Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA.
Nat Protoc. 2018 Oct;13(10):2268-2296. doi: 10.1038/s41596-018-0037-2.
The MS2 system has been widely used, in organisms ranging from bacteria to higher eukaryotes, to image single mRNAs in intact cells with high precision. We have recently re-engineered the MS2 system for accurate detection of mRNAs in living Saccharomyces cerevisiae. Previous MS2 systems affected the degradation of the tagged mRNA, which led to accumulation of MS2 fragments and to erroneous conclusions about mRNA localization and expression. Here we describe a step-by-step protocol for the use of our latest MS2 system (MBSV6) for detecting endogenously tagged mRNAs using wide-field fluorescent microscopy in living yeast. The procedure is divided into three stages: tagging of endogenous gene with MBSV6 (~2 weeks), a two-color single-molecule RNA fluorescent in situ hybridization (smFISH) procedure to quantitatively assess whether mRNAs tagged with MS2 and MS2-coat protein (MCP) behave like untagged mRNAs (2 d, plus additional time for quantification), and a procedure to quantify single mRNAs by live imaging using wide-field microscopy (1 d, plus additional time for quantification). With this method it is now possible to interrogate all phases of mRNA expression, from transcription through decay. The described protocol is designed for S. cerevisiae; however, we think that our approach and the considerations discussed here can be extended to Escherichia coli, Drosophila, Caenorhabditis elegans, and mammalian cells.
MS2 系统已被广泛应用于从细菌到高等真核生物的各种生物体中，以高精度在完整细胞内对单个 mRNA 进行成像。我们最近对 MS2 系统进行了重新设计，以准确检测活酿酒酵母中的 mRNA。以前的 MS2 系统会影响标记 mRNA 的降解，导致 MS2 片段的积累，并导致关于 mRNA 定位和表达的错误结论。在这里，我们描述了使用我们最新的 MS2 系统（MBSV6）在活酵母中使用宽场荧光显微镜检测内源性标记 mRNA 的逐步方案。该过程分为三个阶段：用 MBSV6 对内源基因进行标记（约 2 周）、使用双色单分子 RNA 荧光原位杂交（smFISH）程序定量评估 MS2 和 MS2 衣壳蛋白（MCP）标记的 mRNAs 是否表现得像未标记的 mRNAs（2 天，加上额外的定量时间），以及使用宽场显微镜通过活细胞成像来定量单个 mRNA 的程序（1 天，加上额外的定量时间）。有了这种方法，现在就可以检测 mRNA 表达的所有阶段，从转录到降解。所描述的方案是为酿酒酵母设计的；然而，我们认为我们的方法和这里讨论的考虑因素可以扩展到大肠杆菌、果蝇、秀丽隐杆线虫和哺乳动物细胞。
