Protein-mRNA Interactions Observed in Living Cells By Dual-Color Fluorescence Fluctuation Spectroscopy

Abstract

RNA binding protein controls the localization, translation and degradation of mRNA of specific proteins, therefore exerting a key influence on the cell metabolism, motility and differentiation. The zipcode binding protein 1 (ZBP1) binds to the 3′ untranslated region (UTR) of beta-actin mRNA and regulates its translation. Quantitative characterization of the interaction between mRNA and protein is crucial to dissect this regulation mechanism. We have developed a dual-color fluorescence fluctuation spectroscopy (FFS) technique to study the interaction between mRNA and protein directly in living cells. FFS determines the brightness, concentration and diffusion time of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume. Dual-color FFS distinguishes fluorescent species by brightness and diffusion time as well as the fluorescence color. In this study, we apply dual-color FFS to study the interaction between beta-actin mRNA and ZBP1. The endogenous mouse beta-actin mRNA is visualized by incorporating 24 MS2 stem-loops in the 3′ UTR, which are specifically bound by fluorescently labeled MS2 coat protein (MCP). We have developed a tandem dimeric MCP system that is particularly suitable for FFS brightness analysis. Since multiple MCPs bind to a single mRNA, the brightness of mRNA is significantly higher than that of free MCP, which allows us to readily resolve mRNA from free MCPs. Furthermore, ZBP1 is labeled with a different color and dual-color FFS directly extracts the interacting mRNA-ZBP1 species. The interaction is visualized by brightness signatures and the binding affinity is extracted by measuring different cells with varying concentrations of ZBP1. This work is supported by National Institute of Health EB2060.