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Real time monitoring of endogenous cytoplasmic mRNA using linear antisense 2'-O-methyl RNA probes in living cells.

Okabe K, Harada Y, Zhang J, Tadakuma H, Tani T, Funatsu T - Nucleic Acids Res. (2010)

Bottom Line: Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation.Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA.Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.

ABSTRACT
Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

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Real time monitoring of endogenous c-fos mRNA induction. Normalized FRET fluorescence from streptavidin-bound linear antisense 2′OMeRNA probes was recorded over time. Red line indicates the result from PMA-treated COS7 cells and gray line indicates the result from COS7 cells treated with diluents (0.0001% DMSO). The probe concentration inside the cell was 12 ± 4.8 µM (five cells). Error bars represent SD.
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Figure 5: Real time monitoring of endogenous c-fos mRNA induction. Normalized FRET fluorescence from streptavidin-bound linear antisense 2′OMeRNA probes was recorded over time. Red line indicates the result from PMA-treated COS7 cells and gray line indicates the result from COS7 cells treated with diluents (0.0001% DMSO). The probe concentration inside the cell was 12 ± 4.8 µM (five cells). Error bars represent SD.

Mentions: Linear antisense 2′OMeRNA probe was applied to the real time monitoring of fast-response gene expression. c-fos is an early response gene that is rapidly induced by the stimulation of serum responsive elements (SREs) (24,26,27). Expression of c-Fos protein is upregulated within 1 h upon PMA stimulation, which activates the SRE driven c-Fos promoter (24). In this study, expression of endogenous c-fos mRNA in COS7 cells was monitored by observing FRET fluorescence of linear antisense probes after PMA stimulation. As shown in Figure 5, FRET fluorescence from the cytoplasm of PMA-treated cells began to rise 10 min after the PMA stimulus and a gradual increase continued over time. More than a 6% increase of FRET fluorescence was observed. Nevertheless, we cannot convert this value into the actual increased quantity of induced target mRNA because there is no linear relationship between the FRET intensity and the quantity of the target mRNA. FRET efficiency strongly depends on the concentration ratio of antisense probe to mRNA. The addition of trace diluents (0.0001% DMSO) resulted in a slight decrease of the signal. When linear sense probes were used, no FRET signal higher (defined by FFRET divided by Fdonor) than background noise was observed at any time point. These results indicated that rapid changes in endogenous mRNA expression can be monitored in real time using linear antisense probes by virtue of their fast hybridization kinetics.Figure 5.


Real time monitoring of endogenous cytoplasmic mRNA using linear antisense 2'-O-methyl RNA probes in living cells.

Okabe K, Harada Y, Zhang J, Tadakuma H, Tani T, Funatsu T - Nucleic Acids Res. (2010)

Real time monitoring of endogenous c-fos mRNA induction. Normalized FRET fluorescence from streptavidin-bound linear antisense 2′OMeRNA probes was recorded over time. Red line indicates the result from PMA-treated COS7 cells and gray line indicates the result from COS7 cells treated with diluents (0.0001% DMSO). The probe concentration inside the cell was 12 ± 4.8 µM (five cells). Error bars represent SD.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045578&req=5

Figure 5: Real time monitoring of endogenous c-fos mRNA induction. Normalized FRET fluorescence from streptavidin-bound linear antisense 2′OMeRNA probes was recorded over time. Red line indicates the result from PMA-treated COS7 cells and gray line indicates the result from COS7 cells treated with diluents (0.0001% DMSO). The probe concentration inside the cell was 12 ± 4.8 µM (five cells). Error bars represent SD.
Mentions: Linear antisense 2′OMeRNA probe was applied to the real time monitoring of fast-response gene expression. c-fos is an early response gene that is rapidly induced by the stimulation of serum responsive elements (SREs) (24,26,27). Expression of c-Fos protein is upregulated within 1 h upon PMA stimulation, which activates the SRE driven c-Fos promoter (24). In this study, expression of endogenous c-fos mRNA in COS7 cells was monitored by observing FRET fluorescence of linear antisense probes after PMA stimulation. As shown in Figure 5, FRET fluorescence from the cytoplasm of PMA-treated cells began to rise 10 min after the PMA stimulus and a gradual increase continued over time. More than a 6% increase of FRET fluorescence was observed. Nevertheless, we cannot convert this value into the actual increased quantity of induced target mRNA because there is no linear relationship between the FRET intensity and the quantity of the target mRNA. FRET efficiency strongly depends on the concentration ratio of antisense probe to mRNA. The addition of trace diluents (0.0001% DMSO) resulted in a slight decrease of the signal. When linear sense probes were used, no FRET signal higher (defined by FFRET divided by Fdonor) than background noise was observed at any time point. These results indicated that rapid changes in endogenous mRNA expression can be monitored in real time using linear antisense probes by virtue of their fast hybridization kinetics.Figure 5.

Bottom Line: Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation.Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA.Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.

ABSTRACT
Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.

Show MeSH