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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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Duplex specificity of siRNAs for complex formation. (A) Duplex requirement for complex formation. Sense (S*) or antisense (AS*) strand of EGFPS1A siRNA was incubated separately in the cell extract, and their gel shift patterns were compared with the intact EGFPS1A duplex. The duplex specificity of the complex was further tested with EGFPS1 A siRNA containing either one (S*/AS or S/AS*) or both (S*/AS*) 5′-labeled strands (lanes 3–6). Cold S/AS was added as a competitor (lane 4). Both S*/AS and S/AS* EGFPS1A were incorporated into the complex (arrow).
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Figure 2: Duplex specificity of siRNAs for complex formation. (A) Duplex requirement for complex formation. Sense (S*) or antisense (AS*) strand of EGFPS1A siRNA was incubated separately in the cell extract, and their gel shift patterns were compared with the intact EGFPS1A duplex. The duplex specificity of the complex was further tested with EGFPS1 A siRNA containing either one (S*/AS or S/AS*) or both (S*/AS*) 5′-labeled strands (lanes 3–6). Cold S/AS was added as a competitor (lane 4). Both S*/AS and S/AS* EGFPS1A were incorporated into the complex (arrow).

Mentions: We next tested the specificity of complex formation with single- or double-stranded siRNAs to determine if the high-molecular weight complex observed is an RNA duplex specific (Figure 2). When either the EGFPS1A labeled sense (S*) or antisense (AS*) strands were tested individually in the binding assays, no binding to the high-molecular weight complex was observed (Figure 2) whereas a faster migrating complex was observed with both of the single-strand RNAs. We further tested the duplex requirement of the complex using EGFPS1A containing either one (S*/AS or S/AS*) or both (S*/AS*) 5′-radiolabeled strands. Both S*/AS and S/AS* were incorporated into the complex, and cold EGFPS1A (S/AS) efficiently competed with the S*/AS* siRNA–protein complex, confirming that the complex in this study is duplex specific (Figure 2a). These observations demonstrate that the complex formation in this study is preferentially with the duplex form of siRNAs. Our observations are also consistent with reported observations that the human RLC forms exclusively with the duplex form of siRNAs (27–29).Figure 2.


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)

Duplex specificity of siRNAs for complex formation. (A) Duplex requirement for complex formation. Sense (S*) or antisense (AS*) strand of EGFPS1A siRNA was incubated separately in the cell extract, and their gel shift patterns were compared with the intact EGFPS1A duplex. The duplex specificity of the complex was further tested with EGFPS1 A siRNA containing either one (S*/AS or S/AS*) or both (S*/AS*) 5′-labeled strands (lanes 3–6). Cold S/AS was added as a competitor (lane 4). Both S*/AS and S/AS* EGFPS1A were incorporated into the complex (arrow).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Duplex specificity of siRNAs for complex formation. (A) Duplex requirement for complex formation. Sense (S*) or antisense (AS*) strand of EGFPS1A siRNA was incubated separately in the cell extract, and their gel shift patterns were compared with the intact EGFPS1A duplex. The duplex specificity of the complex was further tested with EGFPS1 A siRNA containing either one (S*/AS or S/AS*) or both (S*/AS*) 5′-labeled strands (lanes 3–6). Cold S/AS was added as a competitor (lane 4). Both S*/AS and S/AS* EGFPS1A were incorporated into the complex (arrow).
Mentions: We next tested the specificity of complex formation with single- or double-stranded siRNAs to determine if the high-molecular weight complex observed is an RNA duplex specific (Figure 2). When either the EGFPS1A labeled sense (S*) or antisense (AS*) strands were tested individually in the binding assays, no binding to the high-molecular weight complex was observed (Figure 2) whereas a faster migrating complex was observed with both of the single-strand RNAs. We further tested the duplex requirement of the complex using EGFPS1A containing either one (S*/AS or S/AS*) or both (S*/AS*) 5′-radiolabeled strands. Both S*/AS and S/AS* were incorporated into the complex, and cold EGFPS1A (S/AS) efficiently competed with the S*/AS* siRNA–protein complex, confirming that the complex in this study is duplex specific (Figure 2a). These observations demonstrate that the complex formation in this study is preferentially with the duplex form of siRNAs. Our observations are also consistent with reported observations that the human RLC forms exclusively with the duplex form of siRNAs (27–29).Figure 2.

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