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A role for human Dicer in pre-RISC loading of siRNAs.

Sakurai K, Amarzguioui M, Kim DH, Alluin J, Heale B, Song MS, Gatignol A, Behlke MA, Rossi JJ - Nucleic Acids Res. (2010)

Bottom Line: Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer.We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3' overhangs and the thermodynamic properties of 2-4 bp on both ends of effective siRNAs.Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, 1450 East Duarte Road, Duarte, CA 91010, USA.

ABSTRACT
RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3' overhangs and the thermodynamic properties of 2-4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential of an siRNA.

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Ribonucleoprotein complex formation and EGFPS1 siRNA silencing. (a) A set of 10 anti-EGFP siRNAs-targeting EGFP Site I (40). Upper strand is sense; bottom strand is antisense. (b) Gel-shift assays. Anti-EGFPS1-A to -J siRNAs were incubated with HEK293 cell extract and resolved in a 4.5% non-denaturing gel. The band shifts were quantified as described in Figure 1. The assays were conducted multiple times with similar results, and a representative gel is shown here. (c) Determination of EGFPS1 siRNA IC50 values. HEK293 cells were co-transfected with either psiEGFPS1-Sense or -Antisense reporter plasmid and an siRNA ranging from 0.2 pM to 1.5 nM in serial dilutions (a minimal of 10 points measurements). Cell lysates collected 24-h post-transfection was subjected to the Dual-Luciferase Reporter Assay System (Promega) (dose-dependent target knockdown measurement curves are shown in Supplementary Figure S1). The n.a. indicates knockdown data were not obtainable for the passenger strand of S1J.
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Figure 3: Ribonucleoprotein complex formation and EGFPS1 siRNA silencing. (a) A set of 10 anti-EGFP siRNAs-targeting EGFP Site I (40). Upper strand is sense; bottom strand is antisense. (b) Gel-shift assays. Anti-EGFPS1-A to -J siRNAs were incubated with HEK293 cell extract and resolved in a 4.5% non-denaturing gel. The band shifts were quantified as described in Figure 1. The assays were conducted multiple times with similar results, and a representative gel is shown here. (c) Determination of EGFPS1 siRNA IC50 values. HEK293 cells were co-transfected with either psiEGFPS1-Sense or -Antisense reporter plasmid and an siRNA ranging from 0.2 pM to 1.5 nM in serial dilutions (a minimal of 10 points measurements). Cell lysates collected 24-h post-transfection was subjected to the Dual-Luciferase Reporter Assay System (Promega) (dose-dependent target knockdown measurement curves are shown in Supplementary Figure S1). The n.a. indicates knockdown data were not obtainable for the passenger strand of S1J.

Mentions: All siRNAs used in this study were synthesized by IDT (Coralville, Iowa). The siRNAs were purified using high-performance liquid chromatography. The sequences are listed in Figures 1 and 3.Figure 1.


A role for human Dicer in pre-RISC loading of siRNAs.

Sakurai K, Amarzguioui M, Kim DH, Alluin J, Heale B, Song MS, Gatignol A, Behlke MA, Rossi JJ - Nucleic Acids Res. (2010)

Ribonucleoprotein complex formation and EGFPS1 siRNA silencing. (a) A set of 10 anti-EGFP siRNAs-targeting EGFP Site I (40). Upper strand is sense; bottom strand is antisense. (b) Gel-shift assays. Anti-EGFPS1-A to -J siRNAs were incubated with HEK293 cell extract and resolved in a 4.5% non-denaturing gel. The band shifts were quantified as described in Figure 1. The assays were conducted multiple times with similar results, and a representative gel is shown here. (c) Determination of EGFPS1 siRNA IC50 values. HEK293 cells were co-transfected with either psiEGFPS1-Sense or -Antisense reporter plasmid and an siRNA ranging from 0.2 pM to 1.5 nM in serial dilutions (a minimal of 10 points measurements). Cell lysates collected 24-h post-transfection was subjected to the Dual-Luciferase Reporter Assay System (Promega) (dose-dependent target knockdown measurement curves are shown in Supplementary Figure S1). The n.a. indicates knockdown data were not obtainable for the passenger strand of S1J.
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Related In: Results  -  Collection

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Figure 3: Ribonucleoprotein complex formation and EGFPS1 siRNA silencing. (a) A set of 10 anti-EGFP siRNAs-targeting EGFP Site I (40). Upper strand is sense; bottom strand is antisense. (b) Gel-shift assays. Anti-EGFPS1-A to -J siRNAs were incubated with HEK293 cell extract and resolved in a 4.5% non-denaturing gel. The band shifts were quantified as described in Figure 1. The assays were conducted multiple times with similar results, and a representative gel is shown here. (c) Determination of EGFPS1 siRNA IC50 values. HEK293 cells were co-transfected with either psiEGFPS1-Sense or -Antisense reporter plasmid and an siRNA ranging from 0.2 pM to 1.5 nM in serial dilutions (a minimal of 10 points measurements). Cell lysates collected 24-h post-transfection was subjected to the Dual-Luciferase Reporter Assay System (Promega) (dose-dependent target knockdown measurement curves are shown in Supplementary Figure S1). The n.a. indicates knockdown data were not obtainable for the passenger strand of S1J.
Mentions: All siRNAs used in this study were synthesized by IDT (Coralville, Iowa). The siRNAs were purified using high-performance liquid chromatography. The sequences are listed in Figures 1 and 3.Figure 1.

Bottom Line: Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer.We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3' overhangs and the thermodynamic properties of 2-4 bp on both ends of effective siRNAs.Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, 1450 East Duarte Road, Duarte, CA 91010, USA.

ABSTRACT
RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3' overhangs and the thermodynamic properties of 2-4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential of an siRNA.

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