title: Imaging mRNA and protein interactions within neurons.
abstract: RNA-protein interactions are essential for proper gene expression regulation, particularly in neurons with unique spatial constraints. Currently, these interactions are defined biochemically, but a method is needed to evaluate them quantitatively within morphological context. Colocalization of two-color labels using wide-field microscopy is a method to infer these interactions. However, because of chromatic aberrations in the objective lens, this approach lacks the resolution to determine whether two molecules are physically in contact or simply nearby by chance. Here, we developed a robust super registration methodology that corrected the chromatic aberration across the entire image field to within 10 nm, which is capable of determining whether two molecules are physically interacting or simply in proximity by random chance. We applied this approach to image single-molecule FISH in combination with immunofluorescence (smFISH-IF) and determined whether the association between an mRNA and binding protein(s) within a neuron was significant or accidental. We evaluated several mRNA-binding proteins identified from RNA pulldown assays to determine which of these exhibit bona fide interactions. Surprisingly, many known mRNA-binding proteins did not bind the mRNA in situ, indicating that adventitious interactions are significant using existing technology. This method provides an ability to evaluate two-color registration compatible with the scale of molecular interactions.
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461.
authors.forename: Carolina
authors.initials: C
authors.lastname: Eliscovich
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461.|Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461.
authors.forename: Shailesh M
authors.initials: SM
authors.lastname: Shenoy
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461; robert.singer@einstein.yu.edu.|Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461.|Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA 20147.
authors.forename: Robert H
authors.identifier: 0000-0002-6725-0093
authors.initials: RH
authors.lastname: Singer
mesh_terms: D005456:Fluorescent Dyes / Q000737:chemistry; D005786:Gene Expression Regulation / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D009474:Neurons / Q000737:chemistry* / Q000648:ultrastructure; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D000072760:Single Molecule Imaging / Q000379:methods
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins
keywords: chromatic aberration correction; smFISH-IF; super registration