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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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The real time imaging of endogenous c-fos mRNA in SGs. (A) PC and fluorescence images of endogenous c-fos mRNA and GFP-TIA1 in COS7 cells before and 30 min after arsenite treatment. Endogenous c-fos mRNA was visualized by acquiring FRET fluorescence from streptavidin-bound linear antisense 2′OMeRNA probes. The probe concentration inside the cell was 0.76 ± 0.24 µM. (B) Co-localization of TIA-1 (green) and endogenous c-fos mRNA (magenta) in SGs. Images of cells are enlarged from boxed area in (A). Scale bars, 10 µm.
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Figure 6: The real time imaging of endogenous c-fos mRNA in SGs. (A) PC and fluorescence images of endogenous c-fos mRNA and GFP-TIA1 in COS7 cells before and 30 min after arsenite treatment. Endogenous c-fos mRNA was visualized by acquiring FRET fluorescence from streptavidin-bound linear antisense 2′OMeRNA probes. The probe concentration inside the cell was 0.76 ± 0.24 µM. (B) Co-localization of TIA-1 (green) and endogenous c-fos mRNA (magenta) in SGs. Images of cells are enlarged from boxed area in (A). Scale bars, 10 µm.

Mentions: To demonstrate the feasibility of using linear antisense 2′OMeRNA probes in the observation of rapid and dynamic changes in the intracellular distribution of mRNA, the accumulation of endogenous mRNA in SGs was imaged. Real time imaging of RNA-binding proteins that reside in SGs and of RNAs, such as poly(A)+ mRNA and viral RNA, revealed that mRNA accumulated in SGs within ∼30 min after the induction of stress with arsenite (18,35). Here, the accumulation of endogenous c-fos mRNA in COS7 cells after sodium arsenite treatment, a conventional stress inducer, was monitored using linear antisense probes. As shown in Figure 6A, FRET fluorescence from stressed cells showed that a fraction of endogenous c-fos mRNA localized in granular domains in the cytoplasm. These granules were confirmed to be SGs by merging the images of c-fos mRNA with those of GFP-TIA-1, an SG marker (Figure 6B). When linear sense probes were used, no significant FRET fluorescence was detected at any time point, demonstrating the mRNA specificity of the linear antisense probe. It is also noteworthy that a fraction of endogenous c-fos mRNA was also detected diffusely throughout the cytoplasm, in contrast to the distribution of TIA-1, which was mostly detected only in SGs and in the nucleus (Figure 6A and B). Even 60 min after stress induction, when the accumulation of mRNA in SGs would have reached saturation, endogenous c-fos mRNA was still detected both in SGs and in the cytoplasm (Supplementary Figure S6). This distribution of a specific endogenous mRNA agrees with a former investigation which showed that only a portion of poly(A)+ mRNAs accumulate in SGs, while a further portion remains in the cytoplasm (35).Figure 6.


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)

The real time imaging of endogenous c-fos mRNA in SGs. (A) PC and fluorescence images of endogenous c-fos mRNA and GFP-TIA1 in COS7 cells before and 30 min after arsenite treatment. Endogenous c-fos mRNA was visualized by acquiring FRET fluorescence from streptavidin-bound linear antisense 2′OMeRNA probes. The probe concentration inside the cell was 0.76 ± 0.24 µM. (B) Co-localization of TIA-1 (green) and endogenous c-fos mRNA (magenta) in SGs. Images of cells are enlarged from boxed area in (A). Scale bars, 10 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: The real time imaging of endogenous c-fos mRNA in SGs. (A) PC and fluorescence images of endogenous c-fos mRNA and GFP-TIA1 in COS7 cells before and 30 min after arsenite treatment. Endogenous c-fos mRNA was visualized by acquiring FRET fluorescence from streptavidin-bound linear antisense 2′OMeRNA probes. The probe concentration inside the cell was 0.76 ± 0.24 µM. (B) Co-localization of TIA-1 (green) and endogenous c-fos mRNA (magenta) in SGs. Images of cells are enlarged from boxed area in (A). Scale bars, 10 µm.
Mentions: To demonstrate the feasibility of using linear antisense 2′OMeRNA probes in the observation of rapid and dynamic changes in the intracellular distribution of mRNA, the accumulation of endogenous mRNA in SGs was imaged. Real time imaging of RNA-binding proteins that reside in SGs and of RNAs, such as poly(A)+ mRNA and viral RNA, revealed that mRNA accumulated in SGs within ∼30 min after the induction of stress with arsenite (18,35). Here, the accumulation of endogenous c-fos mRNA in COS7 cells after sodium arsenite treatment, a conventional stress inducer, was monitored using linear antisense probes. As shown in Figure 6A, FRET fluorescence from stressed cells showed that a fraction of endogenous c-fos mRNA localized in granular domains in the cytoplasm. These granules were confirmed to be SGs by merging the images of c-fos mRNA with those of GFP-TIA-1, an SG marker (Figure 6B). When linear sense probes were used, no significant FRET fluorescence was detected at any time point, demonstrating the mRNA specificity of the linear antisense probe. It is also noteworthy that a fraction of endogenous c-fos mRNA was also detected diffusely throughout the cytoplasm, in contrast to the distribution of TIA-1, which was mostly detected only in SGs and in the nucleus (Figure 6A and B). Even 60 min after stress induction, when the accumulation of mRNA in SGs would have reached saturation, endogenous c-fos mRNA was still detected both in SGs and in the cytoplasm (Supplementary Figure S6). This distribution of a specific endogenous mRNA agrees with a former investigation which showed that only a portion of poly(A)+ mRNAs accumulate in SGs, while a further portion remains in the cytoplasm (35).Figure 6.

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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