title: In vivo imaging of protein-protein and RNA-protein interactions using novel far-red fluorescence complementation systems.
abstract: Imaging of protein-protein and RNA-protein interactions in vivo, especially in live animals, is still challenging. Here we developed far-red mNeptune-based bimolecular fluorescence complementation (BiFC) and trimolecular fluorescence complementation (TriFC) systems with excitation and emission above 600 nm in the 'tissue optical window' for imaging of protein-protein and RNA-protein interactions in live cells and mice. The far-red mNeptune BiFC was first built by selecting appropriate split mNeptune fragments, and then the mNeptune-TriFC system was built based on the mNeptune-BiFC system. The newly constructed mNeptune BiFC and TriFC systems were verified as useful tools for imaging protein-protein and mRNA-protein interactions, respectively, in live cells and mice. We then used the new mNeptune-TriFC system to investigate the interactions between human polypyrimidine-tract-binding protein (PTB) and HIV-1 mRNA elements as PTB may participate in HIV mRNA processing in HIV activation from latency. An interaction between PTB and the 3'long terminal repeat region of HIV-1 mRNAs was found and imaged in live cells and mice, implying a role for PTB in regulating HIV-1 mRNA processing. The study provides new tools for in vivo imaging of RNA-protein and protein-protein interactions, and adds new insight into the mechanism of HIV-1 mRNA processing.
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China Graduate University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Han
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China Graduate University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Shifeng
authors.initials: S
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Zhiping
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Xiaohe
authors.initials: X
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Jiaoyu
authors.initials: J
authors.lastname: Deng
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China.
authors.forename: Hongping
authors.initials: H
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China Graduate University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Zhaoyang
authors.initials: Z
authors.lastname: Li
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China National Key Laboratory of Macrobiomolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China czq@wh.iov.cn.
authors.forename: Xian-En
authors.initials: XE
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China zhangxe@ibp.ac.cn.
authors.forename: Zongqiang
authors.initials: Z
authors.lastname: Cui
mesh_terms: D000818:Animals; D002460:Cell Line; D005470:Fluorometry / Q000379:methods*; D006678:HIV / Q000235:genetics; D016325:HIV Long Terminal Repeat; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D038941:Polypyrimidine Tract-Binding Protein / Q000032:analysis; D025941:Protein Interaction Mapping / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis; D016601:RNA-Binding Proteins / Q000032:analysis*
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D038941:Polypyrimidine Tract-Binding Protein