title: Quantifying Protein-mRNA Interactions in Single Live Cells.
abstract: 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.
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Wu
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Adina R
authors.initials: AR
authors.lastname: Buxbaum
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Zachary B
authors.initials: ZB
authors.lastname: Katz
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Young J
authors.initials: YJ
authors.lastname: Yoon
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA. Electronic address: robert.singer@einstein.yu.edu.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Singer
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000378:metabolism*; D005453:Fluorescence; D006023:Glycoproteins / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D012270:Ribosomes / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D000199:Actins; D006023:Glycoproteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C402632:Zbp1 protein, mouse
keywords: ZBP1; beta-actin mRNA; fluorescence fluctuation spectroscopy; ribosomes; translational regulation