In vivo imaging of protein-protein and RNA-protein interactions using novel far-red fluorescence complementation systems.
Bottom Line: Imaging of protein-protein and RNA-protein interactions in vivo, especially in live animals, is still challenging.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.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.
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.Show MeSH
Related in: MedlinePlus
Mentions: The mNeptune-TriFC system was next used to study unknown protein–mRNA interactions in live cells. Interactions between PTB and the HIV-1 mRNAs were tested using the mNeptune-TriFC system. The 3′LTR (untranslated region) and 5′LTR of HIV-1 mRNAs exist in all synthesized viral mRNAs and play important roles in viral mRNA processing. We assessed the interactions of PTB with the 3′3′LTR and 5′LTR of HIV-1 mRNAs. In the TriFC system, cells co-expressing PTB and the 3′LTR of HIV-1 mRNAs displayed a strong red TriFC signal, while cells co-expressing PTB and the 5′LTR of HIV-1 mRNAs did not produce a red TriFC signal (Figure 5A). The imaging and quantification (Figure 5B) demonstrated that PTB associates with the 3′LTR but not the 5′LTR of HIV-1 mRNA. We used co-immunoprecipitation experiments to verify the interaction between PTB and the 3′LTR of HIV-1 mRNA (Figure 5C). Co-immunoprecipitation experiments also showed there is no interaction between PTB and 5′LTR of HIV-1 mRNA. The known interaction between PTB and the CRS region of HIV-1 env mRNA (16) was also imaged in live cells using the mNeptune-TriFC system (Supplementary Figure S6).
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.