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ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues.

Oomoto I, Suzuki-Hirano A, Umeshima H, Han YW, Yanagisawa H, Carlton P, Harada Y, Kengaku M, Okamoto A, Shimogori T, Wang DO - Nucleic Acids Res. (2015)

Bottom Line: Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks.Time-lapse imaging reveals steady-state stability of these RNA foci and dynamic dissipation of 28S rRNA concentrations upon polymerase I inhibition in native brain tissue.Confirming the validity of this technique in a physiological context, the in vivo RNA labeling did not interfere with the function of target RNA nor cause noticeable cytotoxicity or perturbation of cellular behavior.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.

No MeSH data available.


Related in: MedlinePlus

ECHO-liveFISH imaging of poly(A) RNA foci in living HeLa cells reveals immobility. (A) Fluorescence images of HeLa cells transfected with Cy5-d(T)30 or D514-(U)22. Speckled nuclear fluorescence was readily distinguished in the nuclei of D514-(U)22 transfected cells but not in the Cy5-d(T)30 transfected cells. (B) Time-lapse confocal poly(A) RNA images (LSM780) of HeLa cells transfected with D514-(U)22 (also see Supplementary Video 1). The track-line presentations monitor the center position of individual speckles over time (progressing from blue to red). Diffusion coefficient of each poly(A) speckle was plotted over time. (C) Poly(A) RNA staining in HeLa cells after ECHO-liveFISH imaging. Top, D514-(U)22 fluorescence in transfected and mock-transfected cells. Bottom, after imaging with D514-(U)22, cells were fixed, permeabilized and hybridized with Cy5-d(T)30 probes followed by Cy5 fluorescence imaging. Quantification of mean Cy5 fluorescence intensity at individual speckles (MFI) indicated comparable endogenous poly(A) concentrations at nuclear foci in D514-(U)22-transfected and mock-transfected cells. Scale bars: 20 μm. D514: 514 nm excitation, 517–597 nm detection; Cy5: 633 nm excitation, 638–759 nm detection. 0.264 × 0.264 μm pixel size; 50 μs pixel dwell time.
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Figure 2: ECHO-liveFISH imaging of poly(A) RNA foci in living HeLa cells reveals immobility. (A) Fluorescence images of HeLa cells transfected with Cy5-d(T)30 or D514-(U)22. Speckled nuclear fluorescence was readily distinguished in the nuclei of D514-(U)22 transfected cells but not in the Cy5-d(T)30 transfected cells. (B) Time-lapse confocal poly(A) RNA images (LSM780) of HeLa cells transfected with D514-(U)22 (also see Supplementary Video 1). The track-line presentations monitor the center position of individual speckles over time (progressing from blue to red). Diffusion coefficient of each poly(A) speckle was plotted over time. (C) Poly(A) RNA staining in HeLa cells after ECHO-liveFISH imaging. Top, D514-(U)22 fluorescence in transfected and mock-transfected cells. Bottom, after imaging with D514-(U)22, cells were fixed, permeabilized and hybridized with Cy5-d(T)30 probes followed by Cy5 fluorescence imaging. Quantification of mean Cy5 fluorescence intensity at individual speckles (MFI) indicated comparable endogenous poly(A) concentrations at nuclear foci in D514-(U)22-transfected and mock-transfected cells. Scale bars: 20 μm. D514: 514 nm excitation, 517–597 nm detection; Cy5: 633 nm excitation, 638–759 nm detection. 0.264 × 0.264 μm pixel size; 50 μs pixel dwell time.

Mentions: We next transfected a ribonuclease-resistant poly(A) RNA probe D514-(U)22 (U: 2’O-methyl-uridine) into living HeLa cells (33,34). Upon transfection, discrete speckles of concentrated fluorescence were observed in the nucleus and weak and diffuse fluorescence was observed in the cytoplasm. Transfection of Cy5-d(T)30 however, revealed homogeneous fluorescence distribution in both nucleus and cytoplasm (Figure 2A). An in-cell spectral profile in D514-(U)22 transfected cells identified TO as the sole fluorescence source (Supplementary Figure S2C). The lack of fluorescence in the nucleoli is consistent with previous findings that poly(A) RNA is concentrated at ‘interchromatin granule clusters/nuclear speckles’ (1,35). We then performed time-lapse imaging, which revealed stable fluorescence intensity at each individual poly(A) RNA foci over time (Supplementary Figure S2D). The photostability of D514-(U)22 allowed us to take a 80-frame movie (continuous imaging for a total of 80 s) of transfected HeLa cells and track the mass center of each individual speckle to quantify mobility. As shown in Figure 2B, diffusion coefficient of individual speckles was 0.0004 ± 0.0021 μm2/s throughout the time-lapse imaging, indicating both stability and immobility of poly(A) speckles (Figure 2B and Supplementary Video 1). In order to determine whether the dynamic behavior of endogenous poly(A) RNA was affected by ECHO-liveFISH, we fixed the cells at the end of the experiments and re-probed cells with Cy5-d(T)30. Neither fluorescence intensity or cellular distribution of Cy5-d(T)30 was affected as compared to the vehicle-transfected cells, indicating that expression level and trafficking of poly(A) RNA were not perturbed (Figure 2C). Poly(A) nuclear speckles labeled by D514-U22 were spatially separated from nucleoli where fluorescence of D514-U3 and D514-28S probes concentrated, consistently with the well characterized nuclear locations of these three RNA (Supplementary Figure S2E). Thus, the dynamic imaging of poly(A) RNA in living HeLa cells demonstrates photostability, target-selectivity and usefulness of the probes in monitoring intranuclear RNA foci in living cells.


ECHO-liveFISH: in vivo RNA labeling reveals dynamic regulation of nuclear RNA foci in living tissues.

Oomoto I, Suzuki-Hirano A, Umeshima H, Han YW, Yanagisawa H, Carlton P, Harada Y, Kengaku M, Okamoto A, Shimogori T, Wang DO - Nucleic Acids Res. (2015)

ECHO-liveFISH imaging of poly(A) RNA foci in living HeLa cells reveals immobility. (A) Fluorescence images of HeLa cells transfected with Cy5-d(T)30 or D514-(U)22. Speckled nuclear fluorescence was readily distinguished in the nuclei of D514-(U)22 transfected cells but not in the Cy5-d(T)30 transfected cells. (B) Time-lapse confocal poly(A) RNA images (LSM780) of HeLa cells transfected with D514-(U)22 (also see Supplementary Video 1). The track-line presentations monitor the center position of individual speckles over time (progressing from blue to red). Diffusion coefficient of each poly(A) speckle was plotted over time. (C) Poly(A) RNA staining in HeLa cells after ECHO-liveFISH imaging. Top, D514-(U)22 fluorescence in transfected and mock-transfected cells. Bottom, after imaging with D514-(U)22, cells were fixed, permeabilized and hybridized with Cy5-d(T)30 probes followed by Cy5 fluorescence imaging. Quantification of mean Cy5 fluorescence intensity at individual speckles (MFI) indicated comparable endogenous poly(A) concentrations at nuclear foci in D514-(U)22-transfected and mock-transfected cells. Scale bars: 20 μm. D514: 514 nm excitation, 517–597 nm detection; Cy5: 633 nm excitation, 638–759 nm detection. 0.264 × 0.264 μm pixel size; 50 μs pixel dwell time.
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Figure 2: ECHO-liveFISH imaging of poly(A) RNA foci in living HeLa cells reveals immobility. (A) Fluorescence images of HeLa cells transfected with Cy5-d(T)30 or D514-(U)22. Speckled nuclear fluorescence was readily distinguished in the nuclei of D514-(U)22 transfected cells but not in the Cy5-d(T)30 transfected cells. (B) Time-lapse confocal poly(A) RNA images (LSM780) of HeLa cells transfected with D514-(U)22 (also see Supplementary Video 1). The track-line presentations monitor the center position of individual speckles over time (progressing from blue to red). Diffusion coefficient of each poly(A) speckle was plotted over time. (C) Poly(A) RNA staining in HeLa cells after ECHO-liveFISH imaging. Top, D514-(U)22 fluorescence in transfected and mock-transfected cells. Bottom, after imaging with D514-(U)22, cells were fixed, permeabilized and hybridized with Cy5-d(T)30 probes followed by Cy5 fluorescence imaging. Quantification of mean Cy5 fluorescence intensity at individual speckles (MFI) indicated comparable endogenous poly(A) concentrations at nuclear foci in D514-(U)22-transfected and mock-transfected cells. Scale bars: 20 μm. D514: 514 nm excitation, 517–597 nm detection; Cy5: 633 nm excitation, 638–759 nm detection. 0.264 × 0.264 μm pixel size; 50 μs pixel dwell time.
Mentions: We next transfected a ribonuclease-resistant poly(A) RNA probe D514-(U)22 (U: 2’O-methyl-uridine) into living HeLa cells (33,34). Upon transfection, discrete speckles of concentrated fluorescence were observed in the nucleus and weak and diffuse fluorescence was observed in the cytoplasm. Transfection of Cy5-d(T)30 however, revealed homogeneous fluorescence distribution in both nucleus and cytoplasm (Figure 2A). An in-cell spectral profile in D514-(U)22 transfected cells identified TO as the sole fluorescence source (Supplementary Figure S2C). The lack of fluorescence in the nucleoli is consistent with previous findings that poly(A) RNA is concentrated at ‘interchromatin granule clusters/nuclear speckles’ (1,35). We then performed time-lapse imaging, which revealed stable fluorescence intensity at each individual poly(A) RNA foci over time (Supplementary Figure S2D). The photostability of D514-(U)22 allowed us to take a 80-frame movie (continuous imaging for a total of 80 s) of transfected HeLa cells and track the mass center of each individual speckle to quantify mobility. As shown in Figure 2B, diffusion coefficient of individual speckles was 0.0004 ± 0.0021 μm2/s throughout the time-lapse imaging, indicating both stability and immobility of poly(A) speckles (Figure 2B and Supplementary Video 1). In order to determine whether the dynamic behavior of endogenous poly(A) RNA was affected by ECHO-liveFISH, we fixed the cells at the end of the experiments and re-probed cells with Cy5-d(T)30. Neither fluorescence intensity or cellular distribution of Cy5-d(T)30 was affected as compared to the vehicle-transfected cells, indicating that expression level and trafficking of poly(A) RNA were not perturbed (Figure 2C). Poly(A) nuclear speckles labeled by D514-U22 were spatially separated from nucleoli where fluorescence of D514-U3 and D514-28S probes concentrated, consistently with the well characterized nuclear locations of these three RNA (Supplementary Figure S2E). Thus, the dynamic imaging of poly(A) RNA in living HeLa cells demonstrates photostability, target-selectivity and usefulness of the probes in monitoring intranuclear RNA foci in living cells.

Bottom Line: Here, we describe a high-resolution fluorescence RNA imaging method, ECHO-liveFISH, to label endogenous nuclear RNA in living mice and chicks.Time-lapse imaging reveals steady-state stability of these RNA foci and dynamic dissipation of 28S rRNA concentrations upon polymerase I inhibition in native brain tissue.Confirming the validity of this technique in a physiological context, the in vivo RNA labeling did not interfere with the function of target RNA nor cause noticeable cytotoxicity or perturbation of cellular behavior.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan Graduate School of Biostudies, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan.

No MeSH data available.


Related in: MedlinePlus