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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

Subcellular fractionation of T47D cells to isolate whole cells, cytoplasmic fractions, and nuclear fractions.(A) Scheme of dsRNA transfection and subsequent subcellular fractionation. siRNAs specific for AGO1, AGO2, or TRBP and control dsRNA (siCtrl) were transfected into cells at 25 nM using Lipofectamine RNAiMAX. (B–D) Western blot analysis for Tubulin (cytoplasmic marker), Lamin A/C (nuclear marker), and Calnexin (ER marker) showing the purity of each fraction prepared from siCtrl-, siAGO1-, siAGO2, or siTRBP-treated T47D cells. Equal amount of proteins (25 μg) from each fraction were analyzed by SDS-PAGE. Similar western blot data for MDA-MB-453 cells are presented in Figure S1.
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f1: Subcellular fractionation of T47D cells to isolate whole cells, cytoplasmic fractions, and nuclear fractions.(A) Scheme of dsRNA transfection and subsequent subcellular fractionation. siRNAs specific for AGO1, AGO2, or TRBP and control dsRNA (siCtrl) were transfected into cells at 25 nM using Lipofectamine RNAiMAX. (B–D) Western blot analysis for Tubulin (cytoplasmic marker), Lamin A/C (nuclear marker), and Calnexin (ER marker) showing the purity of each fraction prepared from siCtrl-, siAGO1-, siAGO2, or siTRBP-treated T47D cells. Equal amount of proteins (25 μg) from each fraction were analyzed by SDS-PAGE. Similar western blot data for MDA-MB-453 cells are presented in Figure S1.

Mentions: Accurate investigation into the presence of AGO in human cell nuclei depends on stringent purification to exclude cytoplasmic AGO. This is not a trivial task because AGO can be found in the endoplasmic reticulum (ER)34, an organelle closely associated with the nuclear membrane. We used optimized isolation procedures for this study to separate nuclear and cytoplasmic fractions and minimize contamination from the ER3235 (Fig. 1A).


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)

Subcellular fractionation of T47D cells to isolate whole cells, cytoplasmic fractions, and nuclear fractions.(A) Scheme of dsRNA transfection and subsequent subcellular fractionation. siRNAs specific for AGO1, AGO2, or TRBP and control dsRNA (siCtrl) were transfected into cells at 25 nM using Lipofectamine RNAiMAX. (B–D) Western blot analysis for Tubulin (cytoplasmic marker), Lamin A/C (nuclear marker), and Calnexin (ER marker) showing the purity of each fraction prepared from siCtrl-, siAGO1-, siAGO2, or siTRBP-treated T47D cells. Equal amount of proteins (25 μg) from each fraction were analyzed by SDS-PAGE. Similar western blot data for MDA-MB-453 cells are presented in Figure S1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525381&req=5

f1: Subcellular fractionation of T47D cells to isolate whole cells, cytoplasmic fractions, and nuclear fractions.(A) Scheme of dsRNA transfection and subsequent subcellular fractionation. siRNAs specific for AGO1, AGO2, or TRBP and control dsRNA (siCtrl) were transfected into cells at 25 nM using Lipofectamine RNAiMAX. (B–D) Western blot analysis for Tubulin (cytoplasmic marker), Lamin A/C (nuclear marker), and Calnexin (ER marker) showing the purity of each fraction prepared from siCtrl-, siAGO1-, siAGO2, or siTRBP-treated T47D cells. Equal amount of proteins (25 μg) from each fraction were analyzed by SDS-PAGE. Similar western blot data for MDA-MB-453 cells are presented in Figure S1.
Mentions: Accurate investigation into the presence of AGO in human cell nuclei depends on stringent purification to exclude cytoplasmic AGO. This is not a trivial task because AGO can be found in the endoplasmic reticulum (ER)34, an organelle closely associated with the nuclear membrane. We used optimized isolation procedures for this study to separate nuclear and cytoplasmic fractions and minimize contamination from the ER3235 (Fig. 1A).

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