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

Han Y, Wang S, Zhang Z, Ma X, Li W, Zhang X, Deng J, Wei H, Li Z, Zhang XE, Cui Z - Nucleic Acids Res. (2014)

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.

View Article: PubMed Central - PubMed

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

Imaging the interactions between PTB and the 3′LTR and 5′LTR of HIV-1 mRNAs in live cells. (A) TriFC (mNeptune channel) and ECFP signals of 293T cells co-expressing PTB and the 5′LTR or 3′LTR sequences of HIV mRNAs in the TriFC system. Cells were transfected with plasmids expressing MC156-MCP and PTB-MN155 fusion proteins. The reporter mRNAs contained the ms2 operator and the 5′LTR or 3′LTR sequences of HIV mRNAs. Scale bar: 10 μm. (B) Quantitative analysis of TriFC efficiency for the interactions between PTB and the 5′LTR or 3′LTR sequences of HIV mRNAs. Statistical analysis was based on the fluorescence intensity ratio of TriFC/ECFP. The data are given as the mean + SD (n = 60). Statistical significance was evaluated using a two-tailed Student's t-test. *** indicates significantly different from the control groups without 3′LTR or 5′LTR RNA sequences, P< 0.001. (C) Verification of the interaction between PTB and the 3′LTR of HIV-1 mRNAs. Co-immunoprecipitation (CoIP) assays were performed with cell lysate and anti-PTB antibody or control antibody IgG1. The binding of PTB to the 3′LTR of HIV-1 mRNAs was detected by reverse transcription (RT) followed by PCR amplification using primers specific to the 3′LTR of HIV-1 mRNAs and the housekeeping GAPDH mRNA. No amplification was detected in the ‘No RT’ control samples. CoIP experiments also showed there is no interaction between PTB and 5′LTR of HIV-1 mRNA. Western blotting (WB) was performed to monitor the presence of PTB in the samples used in the CoIP assays.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4117741&req=5

Figure 5: Imaging the interactions between PTB and the 3′LTR and 5′LTR of HIV-1 mRNAs in live cells. (A) TriFC (mNeptune channel) and ECFP signals of 293T cells co-expressing PTB and the 5′LTR or 3′LTR sequences of HIV mRNAs in the TriFC system. Cells were transfected with plasmids expressing MC156-MCP and PTB-MN155 fusion proteins. The reporter mRNAs contained the ms2 operator and the 5′LTR or 3′LTR sequences of HIV mRNAs. Scale bar: 10 μm. (B) Quantitative analysis of TriFC efficiency for the interactions between PTB and the 5′LTR or 3′LTR sequences of HIV mRNAs. Statistical analysis was based on the fluorescence intensity ratio of TriFC/ECFP. The data are given as the mean + SD (n = 60). Statistical significance was evaluated using a two-tailed Student's t-test. *** indicates significantly different from the control groups without 3′LTR or 5′LTR RNA sequences, P< 0.001. (C) Verification of the interaction between PTB and the 3′LTR of HIV-1 mRNAs. Co-immunoprecipitation (CoIP) assays were performed with cell lysate and anti-PTB antibody or control antibody IgG1. The binding of PTB to the 3′LTR of HIV-1 mRNAs was detected by reverse transcription (RT) followed by PCR amplification using primers specific to the 3′LTR of HIV-1 mRNAs and the housekeeping GAPDH mRNA. No amplification was detected in the ‘No RT’ control samples. CoIP experiments also showed there is no interaction between PTB and 5′LTR of HIV-1 mRNA. Western blotting (WB) was performed to monitor the presence of PTB in the samples used in the CoIP assays.

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).


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

Han Y, Wang S, Zhang Z, Ma X, Li W, Zhang X, Deng J, Wei H, Li Z, Zhang XE, Cui Z - Nucleic Acids Res. (2014)

Imaging the interactions between PTB and the 3′LTR and 5′LTR of HIV-1 mRNAs in live cells. (A) TriFC (mNeptune channel) and ECFP signals of 293T cells co-expressing PTB and the 5′LTR or 3′LTR sequences of HIV mRNAs in the TriFC system. Cells were transfected with plasmids expressing MC156-MCP and PTB-MN155 fusion proteins. The reporter mRNAs contained the ms2 operator and the 5′LTR or 3′LTR sequences of HIV mRNAs. Scale bar: 10 μm. (B) Quantitative analysis of TriFC efficiency for the interactions between PTB and the 5′LTR or 3′LTR sequences of HIV mRNAs. Statistical analysis was based on the fluorescence intensity ratio of TriFC/ECFP. The data are given as the mean + SD (n = 60). Statistical significance was evaluated using a two-tailed Student's t-test. *** indicates significantly different from the control groups without 3′LTR or 5′LTR RNA sequences, P< 0.001. (C) Verification of the interaction between PTB and the 3′LTR of HIV-1 mRNAs. Co-immunoprecipitation (CoIP) assays were performed with cell lysate and anti-PTB antibody or control antibody IgG1. The binding of PTB to the 3′LTR of HIV-1 mRNAs was detected by reverse transcription (RT) followed by PCR amplification using primers specific to the 3′LTR of HIV-1 mRNAs and the housekeeping GAPDH mRNA. No amplification was detected in the ‘No RT’ control samples. CoIP experiments also showed there is no interaction between PTB and 5′LTR of HIV-1 mRNA. Western blotting (WB) was performed to monitor the presence of PTB in the samples used in the CoIP assays.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117741&req=5

Figure 5: Imaging the interactions between PTB and the 3′LTR and 5′LTR of HIV-1 mRNAs in live cells. (A) TriFC (mNeptune channel) and ECFP signals of 293T cells co-expressing PTB and the 5′LTR or 3′LTR sequences of HIV mRNAs in the TriFC system. Cells were transfected with plasmids expressing MC156-MCP and PTB-MN155 fusion proteins. The reporter mRNAs contained the ms2 operator and the 5′LTR or 3′LTR sequences of HIV mRNAs. Scale bar: 10 μm. (B) Quantitative analysis of TriFC efficiency for the interactions between PTB and the 5′LTR or 3′LTR sequences of HIV mRNAs. Statistical analysis was based on the fluorescence intensity ratio of TriFC/ECFP. The data are given as the mean + SD (n = 60). Statistical significance was evaluated using a two-tailed Student's t-test. *** indicates significantly different from the control groups without 3′LTR or 5′LTR RNA sequences, P< 0.001. (C) Verification of the interaction between PTB and the 3′LTR of HIV-1 mRNAs. Co-immunoprecipitation (CoIP) assays were performed with cell lysate and anti-PTB antibody or control antibody IgG1. The binding of PTB to the 3′LTR of HIV-1 mRNAs was detected by reverse transcription (RT) followed by PCR amplification using primers specific to the 3′LTR of HIV-1 mRNAs and the housekeeping GAPDH mRNA. No amplification was detected in the ‘No RT’ control samples. CoIP experiments also showed there is no interaction between PTB and 5′LTR of HIV-1 mRNA. Western blotting (WB) was performed to monitor the presence of PTB in the samples used in the CoIP assays.
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).

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.

View Article: PubMed Central - PubMed

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