Quantifying Protein-mRNA Interactions in Single Live Cells

• Published in: Cell Vol. 162; no. 1; pp. 211 - 220
• Main Authors: Wu, Bin, Buxbaum, Adina R., Katz, Zachary B., Yoon, Young J., Singer, Robert H.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02-07-2015
• Subjects: Actins - genetics; Actins - metabolism; Animals; beta-actin mRNA; Cells, Cultured; Embryo, Mammalian - cytology; Fibroblasts - metabolism; Fluorescence; fluorescence fluctuation spectroscopy; Glycoproteins - metabolism; Mice; Neurons - metabolism; ribosomes; Ribosomes - metabolism; RNA, Messenger - metabolism; Single-Cell Analysis - methods; translational regulation; ZBP1; ZBP1; ribosomes; translational regulation; beta-actin mRNA; fluorescence fluctuation spectroscopy
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2015.05.054

Specific binding proteins are crucial for the correct spatiotemporal expression of mRNA. To understand this process, a method is required to characterize RNA-protein interactions in single living cells with subcellular resolution. We combined endogenous single RNA and protein detection with two-photon fluorescence fluctuation analysis to measure the average number of proteins bound to mRNA at specific locations within live cells. We applied this to quantify the known binding of zipcode binding protein 1 (ZBP1) and ribosomes to β-actin mRNA within subcellular compartments of primary fibroblasts and neurons. ZBP1-mRNA binding did not occur in nuclei, contrary to previous conclusions. ZBP1 interaction with β-actin mRNA was enhanced perinuclearly in neurons compared to fibroblasts. Cytoplasmic ZBP1 and ribosome binding to the mRNA were anti-correlated depending on their location in the cell. These measurements support a mechanism whereby ZBP1 inhibits translation of localizing mRNA until its release from the mRNA peripherally, allowing ribosome binding.
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•Two-photon detection of single RNAs binding to individual proteins in live cells•Quantitative measurement of protein number bound to mRNAs in cellular compartments•Spatiotemporal quantification of mRNA association with ZBP1 and ribosomes•ZBP1 represses ribosome binding to β-actin mRNA perinuclearly, but not peripherally
Two-photon detection of single RNAs binding to individual proteins in live cells enables quantitative analysis of binding events in different cellular compartments, unveiling a mechanism for translational control in neurons.