title: Imaging of mRNA-protein interactions in live cells using novel mCherry trimolecular fluorescence complementation systems.
abstract: Live cell imaging of mRNA-protein interactions makes it possible to study posttranscriptional processes of cellular and viral gene expression under physiological conditions. In this study, red color mCherry-based trimolecular fluorescence complementation (TriFC) systems were constructed as new tools for visualizing mRNA-protein interaction in living cells using split mCherry fragments and HIV REV-RRE and TAT-TAR peptide-RNA interaction pairs. The new mCherry TriFC systems were successfully used to image RNA-protein interactions such as that between influenza viral protein NS1 and the 5' UTR of influenza viral mRNAs NS, M, and NP. Upon combination of an mCherry TriFC system with a Venus TriFC system, multiple mRNA-protein interactions could be detected simultaneously in the same cells. Then, the new mCherry TriFC system was used for imaging of interactions between influenza A virus mRNAs and some of adapter proteins in cellular TAP nuclear export pathway in live cells. Adapter proteins Aly and UAP56 were found to associate with three kinds of viral mRNAs. Another adapter protein, splicing factor 9G8, only interacted with intron-containing spliced M2 mRNA. Co-immunoprecipitation assays with influenza A virus-infected cells confirmed these interactions. This study provides long-wavelength-spectrum TriFC systems as new tools for visualizing RNA-protein interactions in live cells and help to understand the nuclear export mechanism of influenza A viral mRNAs.
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China ; Graduate University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Yin
authors.forename: Duanhao
authors.initials: D
authors.lastname: Zhu
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Jinyu
authors.initials: J
authors.lastname: Fan
authors.forename: Dong
authors.initials: D
authors.lastname: Men
authors.forename: Jiaoyu
authors.initials: J
authors.lastname: Deng
authors.forename: Hongping
authors.initials: H
authors.lastname: Wei
authors.forename: Xian-En
authors.initials: XE
authors.lastname: Zhang
authors.forename: Zongqiang
authors.initials: Z
authors.lastname: Cui
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D016325:HIV Long Terminal Repeat / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry / Q000378:metabolism; D054884:Host-Pathogen Interactions; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D057054:Molecular Imaging / Q000379:methods*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism; D000097573:Red Fluorescent Protein; D000086082:Viroporin Proteins
chemical_list: D020121:5' Untranslated Regions; D053487:DEAD-box RNA Helicases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D014157:Transcription Factors; D014763:Viral Matrix Proteins; D017361:Viral Nonstructural Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus; D000097573:Red Fluorescent Protein; C105242:ALYREF protein, human; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins; C514860:rev protein, Human Immunodeficiency Virus-1; C487886:DDX39B protein, human