Sugimoto Shinya, Arita-Morioka Ken-ichi, Mizunoe Yoshimitsu, Yamanaka Kunitoshi, Ogura Teru
Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-Ku, Kumamoto 860-0811, Japan Department of Bacteriology, The Jikei University School of Medicine, Minato-Ku, Tokyo 105-8461, Japan
Department of Molecular Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Chuo-Ku, Kumamoto 860-0811, Japan.
Nucleic Acids Res. 2015 Aug 18;43(14):e92. doi: 10.1093/nar/gkv338. Epub 2015 Apr 16.
The intrinsically stochastic dynamics of mRNA metabolism have important consequences on gene regulation and non-genetic cell-to-cell variability; however, no generally applicable methods exist for studying such stochastic processes quantitatively. Here, we describe the use of the amyloid-binding probe Thioflavin T (ThT) for monitoring RNA metabolism in vitro and in vivo. ThT fluoresced strongly in complex with bacterial total RNA than with genomic DNA. ThT bound purine oligoribonucleotides preferentially over pyrimidine oligoribonucleotides and oligodeoxyribonucleotides. This property enabled quantitative real-time monitoring of poly(A) synthesis and phosphorolysis by polyribonucleotide phosphorylase in vitro. Cellular analyses, in combination with genetic approaches and the transcription-inhibitor rifampicin treatment, demonstrated that ThT mainly stained mRNA in actively dividing Escherichia coli cells. ThT also facilitated mRNA metabolism profiling at the single-cell level in diverse bacteria. Furthermore, ThT can also be used to visualise transitions between non-persister and persister cell states, a phenomenon of isogenic subpopulations of antibiotic-sensitive bacteria that acquire tolerance to multiple antibiotics due to stochastically induced dormant states. Collectively, these results suggest that probing mRNA dynamics with ThT is a broadly applicable approach ranging from the molecular level to the single-cell level.
mRNA代谢内在的随机动力学对基因调控和非遗传的细胞间变异性具有重要影响；然而，目前尚无用于定量研究此类随机过程的普遍适用方法。在此，我们描述了使用淀粉样蛋白结合探针硫黄素T（ThT）在体外和体内监测RNA代谢。与基因组DNA相比，ThT与细菌总RNA形成复合物时荧光更强。ThT优先结合嘌呤寡核糖核苷酸，而不是嘧啶寡核糖核苷酸和寡脱氧核糖核苷酸。这一特性使得能够在体外对多聚核苷酸磷酸化酶催化的多聚腺苷酸（poly(A)）合成和磷酸解进行实时定量监测。细胞分析结合遗传方法和转录抑制剂利福平处理表明，ThT主要对活跃分裂的大肠杆菌细胞中的mRNA进行染色。ThT还促进了对多种细菌单细胞水平的mRNA代谢谱分析。此外，ThT还可用于可视化非持久性和持久性细胞状态之间的转变，这是抗生素敏感细菌的同基因亚群中的一种现象，这些细菌由于随机诱导的休眠状态而获得对多种抗生素的耐受性。总体而言，这些结果表明，用ThT探测mRNA动态是一种从分子水平到单细胞水平广泛适用的方法。
