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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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The effect of 3′ overhang on complex formation and RISC function. (a and b) Four variants of EGFPS1A siRNA differing in the number of nucleotides at 3′-end (a) were evaluated for complex formation competence (b) + 2, + 1, + 0, − 1, −2 indicate numbers of nucleotides at the 3′-end as overhangs [19 + 2, 20 + 1, 21 + 0 (blunt ends), 20 – 1 (with 1-nt 5′ overhangs) and 19 – 2 (with 2-nt 5′ overhangs)]. The bottom strands are the antisense sequences and the upper strands the sense sequences. Without HEK293 whole-cell extract, 19 + 2 was included as a negative control. The band shifts were quantified by densitometric scanning of the gel (Typhoon scanner) and percent bound siRNAs were calculated by [bound/(free + bound) × 100] of siRNAs relative to input siRNA without cell-extract incubation. The assays were conducted multiple times with similar results, and a representative gel is shown here. (c) Dual Luciferase assays. To determine the efficiency of the 3′-end modified siRNAs in intracellular target knockdown efficiency, silencing by the antisense strand of the end modified EGFPS1A siRNA duplexes was assayed in HEK293 cells by co-transfecting the psi-EGFP-S1 sense reporter and 20- or 200-pM siRNAs. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections).
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Figure 1: The effect of 3′ overhang on complex formation and RISC function. (a and b) Four variants of EGFPS1A siRNA differing in the number of nucleotides at 3′-end (a) were evaluated for complex formation competence (b) + 2, + 1, + 0, − 1, −2 indicate numbers of nucleotides at the 3′-end as overhangs [19 + 2, 20 + 1, 21 + 0 (blunt ends), 20 – 1 (with 1-nt 5′ overhangs) and 19 – 2 (with 2-nt 5′ overhangs)]. The bottom strands are the antisense sequences and the upper strands the sense sequences. Without HEK293 whole-cell extract, 19 + 2 was included as a negative control. The band shifts were quantified by densitometric scanning of the gel (Typhoon scanner) and percent bound siRNAs were calculated by [bound/(free + bound) × 100] of siRNAs relative to input siRNA without cell-extract incubation. The assays were conducted multiple times with similar results, and a representative gel is shown here. (c) Dual Luciferase assays. To determine the efficiency of the 3′-end modified siRNAs in intracellular target knockdown efficiency, silencing by the antisense strand of the end modified EGFPS1A siRNA duplexes was assayed in HEK293 cells by co-transfecting the psi-EGFP-S1 sense reporter and 20- or 200-pM siRNAs. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections).

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)

The effect of 3′ overhang on complex formation and RISC function. (a and b) Four variants of EGFPS1A siRNA differing in the number of nucleotides at 3′-end (a) were evaluated for complex formation competence (b) + 2, + 1, + 0, − 1, −2 indicate numbers of nucleotides at the 3′-end as overhangs [19 + 2, 20 + 1, 21 + 0 (blunt ends), 20 – 1 (with 1-nt 5′ overhangs) and 19 – 2 (with 2-nt 5′ overhangs)]. The bottom strands are the antisense sequences and the upper strands the sense sequences. Without HEK293 whole-cell extract, 19 + 2 was included as a negative control. The band shifts were quantified by densitometric scanning of the gel (Typhoon scanner) and percent bound siRNAs were calculated by [bound/(free + bound) × 100] of siRNAs relative to input siRNA without cell-extract incubation. The assays were conducted multiple times with similar results, and a representative gel is shown here. (c) Dual Luciferase assays. To determine the efficiency of the 3′-end modified siRNAs in intracellular target knockdown efficiency, silencing by the antisense strand of the end modified EGFPS1A siRNA duplexes was assayed in HEK293 cells by co-transfecting the psi-EGFP-S1 sense reporter and 20- or 200-pM siRNAs. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045585&req=5

Figure 1: The effect of 3′ overhang on complex formation and RISC function. (a and b) Four variants of EGFPS1A siRNA differing in the number of nucleotides at 3′-end (a) were evaluated for complex formation competence (b) + 2, + 1, + 0, − 1, −2 indicate numbers of nucleotides at the 3′-end as overhangs [19 + 2, 20 + 1, 21 + 0 (blunt ends), 20 – 1 (with 1-nt 5′ overhangs) and 19 – 2 (with 2-nt 5′ overhangs)]. The bottom strands are the antisense sequences and the upper strands the sense sequences. Without HEK293 whole-cell extract, 19 + 2 was included as a negative control. The band shifts were quantified by densitometric scanning of the gel (Typhoon scanner) and percent bound siRNAs were calculated by [bound/(free + bound) × 100] of siRNAs relative to input siRNA without cell-extract incubation. The assays were conducted multiple times with similar results, and a representative gel is shown here. (c) Dual Luciferase assays. To determine the efficiency of the 3′-end modified siRNAs in intracellular target knockdown efficiency, silencing by the antisense strand of the end modified EGFPS1A siRNA duplexes was assayed in HEK293 cells by co-transfecting the psi-EGFP-S1 sense reporter and 20- or 200-pM siRNAs. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections).
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.

Show MeSH