In vivo imaging of protein-protein and RNA-protein interactions using novel far-red fluorescence complementation systems

Nucleic Acids Res. 2014 Jul;42(13):e103. doi: 10.1093/nar/gku408. Epub 2014 May 9.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Fluorometry / methods*
  • HIV / genetics
  • HIV Long Terminal Repeat
  • HeLa Cells
  • Humans
  • Mice
  • Microscopy, Fluorescence
  • Polypyrimidine Tract-Binding Protein / analysis
  • Protein Interaction Mapping / methods*
  • RNA, Messenger / analysis*
  • RNA, Viral / analysis
  • RNA-Binding Proteins / analysis*


  • RNA, Messenger
  • RNA, Viral
  • RNA-Binding Proteins
  • Polypyrimidine Tract-Binding Protein