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Reduced Expression of Argonaute 1, Argonaute 2, and TRBP Changes Levels and Intracellular Distribution of RNAi Factors.

Matsui M, Li L, Janowski BA, Corey DR - Sci Rep (2015)

Bottom Line: It is now becoming appreciated that RNAi factors can also be found in cell nuclei, but much remains to be learned about their transport, molecular recognition, and function.We find that siRNA-mediated reduction of AGO1 or AGO2 increases the proportion of AGO1 or AGO2 in cell nuclei.These data reveal the expression of RNAi proteins is mutually dependent and that perturbation can affect subcellular distribution of those factors inside cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, 75390-9041.

ABSTRACT
Until recently, Argonaute 2 (AGO2) and other RNA factors were believed to be restricted to the cytoplasm of mammalian somatic cells. It is now becoming appreciated that RNAi factors can also be found in cell nuclei, but much remains to be learned about their transport, molecular recognition, and function. We find that siRNA-mediated reduction of AGO1 or AGO2 increases the proportion of AGO1 or AGO2 in cell nuclei. Inhibition of AGO1 expression led to increased AGO2 levels, while knockdown of AGO2 led to increased levels of AGO1. Blocking AGO1, AGO2, or TRBP expression changed expression levels and nuclear distribution of RNAi factors Dicer, TNRC6A (GW182), and TRBP. These data reveal the expression of RNAi proteins is mutually dependent and that perturbation can affect subcellular distribution of those factors inside cells.

No MeSH data available.


Related in: MedlinePlus

Immunofluorescence microscopy reveals nuclear retention of AGO2 after AGO2 depletion by siRNA.(A) Immunofluorescence microscopic images for AGO2 in T47D cells after siCtrl transfection. [siCtrl] = 25 nM. (B) Immunofluorescence microscopic images for AGO2 in T47D cells after siAGO2 transfection. [siAGO2] = 25 nM. The images were taken 3 days after transfection of duplex RNAs. Z-sections in the middle of the cells (10 image slices, interval: 0.2 μm) were stacked and projected three-dimensionally. left: AGO2 (green); middle: DAPI (blue) and AGO2 (green); right: cytoplasmic AGO2 (green) and nuclear AGO2 (red). Using Imaris program, fluorescence signals which overlap DAPI’s blue signals are shown in red. The scale bar = 10 μm. See also Figure S6 for additional images. (C) Ratio of fluorescence intensity per unit area in the nucleus relative to the cytoplasm. n = 16. Error bars are SEM. ***p < 0.001 (unpaired t-test).
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f5: Immunofluorescence microscopy reveals nuclear retention of AGO2 after AGO2 depletion by siRNA.(A) Immunofluorescence microscopic images for AGO2 in T47D cells after siCtrl transfection. [siCtrl] = 25 nM. (B) Immunofluorescence microscopic images for AGO2 in T47D cells after siAGO2 transfection. [siAGO2] = 25 nM. The images were taken 3 days after transfection of duplex RNAs. Z-sections in the middle of the cells (10 image slices, interval: 0.2 μm) were stacked and projected three-dimensionally. left: AGO2 (green); middle: DAPI (blue) and AGO2 (green); right: cytoplasmic AGO2 (green) and nuclear AGO2 (red). Using Imaris program, fluorescence signals which overlap DAPI’s blue signals are shown in red. The scale bar = 10 μm. See also Figure S6 for additional images. (C) Ratio of fluorescence intensity per unit area in the nucleus relative to the cytoplasm. n = 16. Error bars are SEM. ***p < 0.001 (unpaired t-test).

Mentions: To further investigate the nuclear retention of AGO2 inside cells we used wide-field immunofluorescence microscopy. After cells were treated with anti-AGO2 siRNA or a noncomplementary control RNA (siCtrl), cells were fixed and then immunostained using anti-AGO2 antibody to examine subcellular distribution of AGO2. Microscopic observation of siCtrl-treated cells revealed AGO2 distributed throughout both cytoplasm and nucleus (Fig. 5A and S6A), consistent with the previous report for T47D cells that had not been transfected with any siRNA32.


Reduced Expression of Argonaute 1, Argonaute 2, and TRBP Changes Levels and Intracellular Distribution of RNAi Factors.

Matsui M, Li L, Janowski BA, Corey DR - Sci Rep (2015)

Immunofluorescence microscopy reveals nuclear retention of AGO2 after AGO2 depletion by siRNA.(A) Immunofluorescence microscopic images for AGO2 in T47D cells after siCtrl transfection. [siCtrl] = 25 nM. (B) Immunofluorescence microscopic images for AGO2 in T47D cells after siAGO2 transfection. [siAGO2] = 25 nM. The images were taken 3 days after transfection of duplex RNAs. Z-sections in the middle of the cells (10 image slices, interval: 0.2 μm) were stacked and projected three-dimensionally. left: AGO2 (green); middle: DAPI (blue) and AGO2 (green); right: cytoplasmic AGO2 (green) and nuclear AGO2 (red). Using Imaris program, fluorescence signals which overlap DAPI’s blue signals are shown in red. The scale bar = 10 μm. See also Figure S6 for additional images. (C) Ratio of fluorescence intensity per unit area in the nucleus relative to the cytoplasm. n = 16. Error bars are SEM. ***p < 0.001 (unpaired t-test).
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Related In: Results  -  Collection

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f5: Immunofluorescence microscopy reveals nuclear retention of AGO2 after AGO2 depletion by siRNA.(A) Immunofluorescence microscopic images for AGO2 in T47D cells after siCtrl transfection. [siCtrl] = 25 nM. (B) Immunofluorescence microscopic images for AGO2 in T47D cells after siAGO2 transfection. [siAGO2] = 25 nM. The images were taken 3 days after transfection of duplex RNAs. Z-sections in the middle of the cells (10 image slices, interval: 0.2 μm) were stacked and projected three-dimensionally. left: AGO2 (green); middle: DAPI (blue) and AGO2 (green); right: cytoplasmic AGO2 (green) and nuclear AGO2 (red). Using Imaris program, fluorescence signals which overlap DAPI’s blue signals are shown in red. The scale bar = 10 μm. See also Figure S6 for additional images. (C) Ratio of fluorescence intensity per unit area in the nucleus relative to the cytoplasm. n = 16. Error bars are SEM. ***p < 0.001 (unpaired t-test).
Mentions: To further investigate the nuclear retention of AGO2 inside cells we used wide-field immunofluorescence microscopy. After cells were treated with anti-AGO2 siRNA or a noncomplementary control RNA (siCtrl), cells were fixed and then immunostained using anti-AGO2 antibody to examine subcellular distribution of AGO2. Microscopic observation of siCtrl-treated cells revealed AGO2 distributed throughout both cytoplasm and nucleus (Fig. 5A and S6A), consistent with the previous report for T47D cells that had not been transfected with any siRNA32.

Bottom Line: It is now becoming appreciated that RNAi factors can also be found in cell nuclei, but much remains to be learned about their transport, molecular recognition, and function.We find that siRNA-mediated reduction of AGO1 or AGO2 increases the proportion of AGO1 or AGO2 in cell nuclei.These data reveal the expression of RNAi proteins is mutually dependent and that perturbation can affect subcellular distribution of those factors inside cells.

View Article: PubMed Central - PubMed

Affiliation: Departments of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, 75390-9041.

ABSTRACT
Until recently, Argonaute 2 (AGO2) and other RNA factors were believed to be restricted to the cytoplasm of mammalian somatic cells. It is now becoming appreciated that RNAi factors can also be found in cell nuclei, but much remains to be learned about their transport, molecular recognition, and function. We find that siRNA-mediated reduction of AGO1 or AGO2 increases the proportion of AGO1 or AGO2 in cell nuclei. Inhibition of AGO1 expression led to increased AGO2 levels, while knockdown of AGO2 led to increased levels of AGO1. Blocking AGO1, AGO2, or TRBP expression changed expression levels and nuclear distribution of RNAi factors Dicer, TNRC6A (GW182), and TRBP. These data reveal the expression of RNAi proteins is mutually dependent and that perturbation can affect subcellular distribution of those factors inside cells.

No MeSH data available.


Related in: MedlinePlus