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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

Effect of AGO1 knockdown on levels of other RNAi factors in the cytoplasm and the nucleus.(A) Western blot data showing levels of Dicer, TNRC6A, AGO1, AGO2, and TRBP proteins in the whole cell and the cytoplasmic and the nuclear fractions after siCtrl or siAGO1 treatment. siCtrl and siAGO1 were transfected into T47D cells at 25 nM. Equal amount of proteins (25 μg) from each fraction were analyzed by SDS-PAGE. Data were representative from three independent experiments. (B) %AGO1 remaining after siAGO1 treatment in the cytoplasm and the nucleus. n = 3. Error bars are SD. **p < 0.01 (unpaired t-test). (C,D) Quantitation of data shown in panel A and independent replicates (n = 3) showing levels of Dicer, TNRC6A, AGO1, AGO2, and TRBP proteins in the cytoplasmic (C) and the nuclear (D) fractions after siAGO1 treatment relative to siCtrl treatment. Error bars are SD. *p < 0.05, **p < 0.01, and ***p < 0.001 relative to siCtrl treatment (paired t-test).
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f2: Effect of AGO1 knockdown on levels of other RNAi factors in the cytoplasm and the nucleus.(A) Western blot data showing levels of Dicer, TNRC6A, AGO1, AGO2, and TRBP proteins in the whole cell and the cytoplasmic and the nuclear fractions after siCtrl or siAGO1 treatment. siCtrl and siAGO1 were transfected into T47D cells at 25 nM. Equal amount of proteins (25 μg) from each fraction were analyzed by SDS-PAGE. Data were representative from three independent experiments. (B) %AGO1 remaining after siAGO1 treatment in the cytoplasm and the nucleus. n = 3. Error bars are SD. **p < 0.01 (unpaired t-test). (C,D) Quantitation of data shown in panel A and independent replicates (n = 3) showing levels of Dicer, TNRC6A, AGO1, AGO2, and TRBP proteins in the cytoplasmic (C) and the nuclear (D) fractions after siAGO1 treatment relative to siCtrl treatment. Error bars are SD. *p < 0.05, **p < 0.01, and ***p < 0.001 relative to siCtrl treatment (paired t-test).

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)

Effect of AGO1 knockdown on levels of other RNAi factors in the cytoplasm and the nucleus.(A) Western blot data showing levels of Dicer, TNRC6A, AGO1, AGO2, and TRBP proteins in the whole cell and the cytoplasmic and the nuclear fractions after siCtrl or siAGO1 treatment. siCtrl and siAGO1 were transfected into T47D cells at 25 nM. Equal amount of proteins (25 μg) from each fraction were analyzed by SDS-PAGE. Data were representative from three independent experiments. (B) %AGO1 remaining after siAGO1 treatment in the cytoplasm and the nucleus. n = 3. Error bars are SD. **p < 0.01 (unpaired t-test). (C,D) Quantitation of data shown in panel A and independent replicates (n = 3) showing levels of Dicer, TNRC6A, AGO1, AGO2, and TRBP proteins in the cytoplasmic (C) and the nuclear (D) fractions after siAGO1 treatment relative to siCtrl treatment. Error bars are SD. *p < 0.05, **p < 0.01, and ***p < 0.001 relative to siCtrl treatment (paired t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Effect of AGO1 knockdown on levels of other RNAi factors in the cytoplasm and the nucleus.(A) Western blot data showing levels of Dicer, TNRC6A, AGO1, AGO2, and TRBP proteins in the whole cell and the cytoplasmic and the nuclear fractions after siCtrl or siAGO1 treatment. siCtrl and siAGO1 were transfected into T47D cells at 25 nM. Equal amount of proteins (25 μg) from each fraction were analyzed by SDS-PAGE. Data were representative from three independent experiments. (B) %AGO1 remaining after siAGO1 treatment in the cytoplasm and the nucleus. n = 3. Error bars are SD. **p < 0.01 (unpaired t-test). (C,D) Quantitation of data shown in panel A and independent replicates (n = 3) showing levels of Dicer, TNRC6A, AGO1, AGO2, and TRBP proteins in the cytoplasmic (C) and the nuclear (D) fractions after siAGO1 treatment relative to siCtrl treatment. Error bars are SD. *p < 0.05, **p < 0.01, and ***p < 0.001 relative to siCtrl treatment (paired t-test).
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