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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

Relative levels of RNAi factors between the cytoplasm and the nucleus in T47D cells.(A) Western blot data showing relative levels of Dicer, TNRC6A, AGO1, AGO2, and TRBP between the cytoplasm and the nucleus in siCtrl-, siAGO1, siAGO2, or siTRBP-treated cells. [dsRNA] = 25 nM. The cytoplasmic and nuclear fractions were prepared in the equal amount of buffers. A equal volume of samples from each fraction were analyzed by SDS-PAGE to evaluate relative levels of each protein between the cytoplasm and the nucleus. Data were representative from three independent experiments. (B) Ratio of each RNAi factor between the cytoplasm and the nucleus after siCtrl, siAGO1, siAGO2, or siTRBP treatment. Data were averaged from 9 data sets for siCtrl-treated samples and 3 data sets for siAGO1-, siAGO2-, or siTRBP-treated samples.
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f3: Relative levels of RNAi factors between the cytoplasm and the nucleus in T47D cells.(A) Western blot data showing relative levels of Dicer, TNRC6A, AGO1, AGO2, and TRBP between the cytoplasm and the nucleus in siCtrl-, siAGO1, siAGO2, or siTRBP-treated cells. [dsRNA] = 25 nM. The cytoplasmic and nuclear fractions were prepared in the equal amount of buffers. A equal volume of samples from each fraction were analyzed by SDS-PAGE to evaluate relative levels of each protein between the cytoplasm and the nucleus. Data were representative from three independent experiments. (B) Ratio of each RNAi factor between the cytoplasm and the nucleus after siCtrl, siAGO1, siAGO2, or siTRBP treatment. Data were averaged from 9 data sets for siCtrl-treated samples and 3 data sets for siAGO1-, siAGO2-, or siTRBP-treated samples.

Mentions: When siRNAs are used to reduce expression of cellular proteins the efficiency of knockdown is typically evaluated by examining preparations obtained from whole cells. One shortcoming of these analyses is that they do not reveal whether reduced protein levels are more pronounced in one cellular compartment relative to another. In addition, most studies focus on evaluating levels of the RNAi factor targeted by the siRNA and do not examine the possibility that expression of other RNAi factors may also change. To accomplish a broader evaluation of the response of RNAi factors to RNAi-mediated gene knockdown of key components of RISC, we examined how using siRNAs to reduce AGO1 expression affected subcellular distribution of AGO1, AGO2, Dicer, TNRC6A, and TRBP proteins (Figs 2 and 3).


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)

Relative levels of RNAi factors between the cytoplasm and the nucleus in T47D cells.(A) Western blot data showing relative levels of Dicer, TNRC6A, AGO1, AGO2, and TRBP between the cytoplasm and the nucleus in siCtrl-, siAGO1, siAGO2, or siTRBP-treated cells. [dsRNA] = 25 nM. The cytoplasmic and nuclear fractions were prepared in the equal amount of buffers. A equal volume of samples from each fraction were analyzed by SDS-PAGE to evaluate relative levels of each protein between the cytoplasm and the nucleus. Data were representative from three independent experiments. (B) Ratio of each RNAi factor between the cytoplasm and the nucleus after siCtrl, siAGO1, siAGO2, or siTRBP treatment. Data were averaged from 9 data sets for siCtrl-treated samples and 3 data sets for siAGO1-, siAGO2-, or siTRBP-treated samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4525381&req=5

f3: Relative levels of RNAi factors between the cytoplasm and the nucleus in T47D cells.(A) Western blot data showing relative levels of Dicer, TNRC6A, AGO1, AGO2, and TRBP between the cytoplasm and the nucleus in siCtrl-, siAGO1, siAGO2, or siTRBP-treated cells. [dsRNA] = 25 nM. The cytoplasmic and nuclear fractions were prepared in the equal amount of buffers. A equal volume of samples from each fraction were analyzed by SDS-PAGE to evaluate relative levels of each protein between the cytoplasm and the nucleus. Data were representative from three independent experiments. (B) Ratio of each RNAi factor between the cytoplasm and the nucleus after siCtrl, siAGO1, siAGO2, or siTRBP treatment. Data were averaged from 9 data sets for siCtrl-treated samples and 3 data sets for siAGO1-, siAGO2-, or siTRBP-treated samples.
Mentions: When siRNAs are used to reduce expression of cellular proteins the efficiency of knockdown is typically evaluated by examining preparations obtained from whole cells. One shortcoming of these analyses is that they do not reveal whether reduced protein levels are more pronounced in one cellular compartment relative to another. In addition, most studies focus on evaluating levels of the RNAi factor targeted by the siRNA and do not examine the possibility that expression of other RNAi factors may also change. To accomplish a broader evaluation of the response of RNAi factors to RNAi-mediated gene knockdown of key components of RISC, we examined how using siRNAs to reduce AGO1 expression affected subcellular distribution of AGO1, AGO2, Dicer, TNRC6A, and TRBP proteins (Figs 2 and 3).

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