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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 for TRBP after control siRNA or siTRBP treatment.(A) Immunofluorescence microscopic images for TRBP in T47D cells after siCtrl transfection. [siCtrl] = 25 nM. (B) Immunofluorescence microscopic images for TRBP in T47D cells after siTRBP transfection. [siTRBP] = 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: TRBP (green); middle: DAPI (blue) and TRBP (green); right: cytoplasmic TRBP (green) and nuclear TRBP (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 = 25. Error bars are SEM. ***p < 0.001 (unpaired t-test).
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f7: Immunofluorescence microscopy for TRBP after control siRNA or siTRBP treatment.(A) Immunofluorescence microscopic images for TRBP in T47D cells after siCtrl transfection. [siCtrl] = 25 nM. (B) Immunofluorescence microscopic images for TRBP in T47D cells after siTRBP transfection. [siTRBP] = 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: TRBP (green); middle: DAPI (blue) and TRBP (green); right: cytoplasmic TRBP (green) and nuclear TRBP (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 = 25. Error bars are SEM. ***p < 0.001 (unpaired t-test).

Mentions: We performed immunofluorescence microscopy as an independent method to evaluate the effect of silencing TRBP on nuclear and cytoplasmic localization of residual protein. When T47D cells were treated with control siRNA we observed distribution of TRBP in the cytoplasm and nucleus, consistent with our western blot analysis (Figs 7A and S6C). We then examined localization of residual TRBP after transfection of an siRNA targeting TRBP. In contrast to the relative enhancement of nuclear AGO2 observed after knockdown of AGO2 (Fig. 5) we observed a modest decrease in the relative levels of nuclear versus cytoplasmic TRBP (Figs 7B,C and S6D). These data show that enhanced nuclear localization is not a universal response to knockdown of RNAi factors.


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 for TRBP after control siRNA or siTRBP treatment.(A) Immunofluorescence microscopic images for TRBP in T47D cells after siCtrl transfection. [siCtrl] = 25 nM. (B) Immunofluorescence microscopic images for TRBP in T47D cells after siTRBP transfection. [siTRBP] = 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: TRBP (green); middle: DAPI (blue) and TRBP (green); right: cytoplasmic TRBP (green) and nuclear TRBP (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 = 25. Error bars are SEM. ***p < 0.001 (unpaired t-test).
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Related In: Results  -  Collection

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f7: Immunofluorescence microscopy for TRBP after control siRNA or siTRBP treatment.(A) Immunofluorescence microscopic images for TRBP in T47D cells after siCtrl transfection. [siCtrl] = 25 nM. (B) Immunofluorescence microscopic images for TRBP in T47D cells after siTRBP transfection. [siTRBP] = 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: TRBP (green); middle: DAPI (blue) and TRBP (green); right: cytoplasmic TRBP (green) and nuclear TRBP (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 = 25. Error bars are SEM. ***p < 0.001 (unpaired t-test).
Mentions: We performed immunofluorescence microscopy as an independent method to evaluate the effect of silencing TRBP on nuclear and cytoplasmic localization of residual protein. When T47D cells were treated with control siRNA we observed distribution of TRBP in the cytoplasm and nucleus, consistent with our western blot analysis (Figs 7A and S6C). We then examined localization of residual TRBP after transfection of an siRNA targeting TRBP. In contrast to the relative enhancement of nuclear AGO2 observed after knockdown of AGO2 (Fig. 5) we observed a modest decrease in the relative levels of nuclear versus cytoplasmic TRBP (Figs 7B,C and S6D). These data show that enhanced nuclear localization is not a universal response to knockdown of RNAi factors.

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