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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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Comparative analyses of guide strand selection for 21-mer and 25/27-mer Dicer substrate siRNAs. Target knockdowns for sense and antisense strands of siRNA EGFPS1A (a) and EGFPS1B (b) in 21-mer and Dicer substrate formats were carried out using co-transfections of the siRNAs with either the psi-EGFP-S sense reporter (red) or psi-EGFP-AS antisense reporter (black) in HEK293 cells. For both targets the 21-nt EGFPS1 siRNAs (left) or 25/27 DsiRNAs (right) were tested at various concentrations. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections). Sequences and IC50 values of each siRNA are shown. For the Dicer substrate siRNAs, the lower case letters represent deoxyribonucleotide containing bases which block Dicer entry.
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Figure 10: Comparative analyses of guide strand selection for 21-mer and 25/27-mer Dicer substrate siRNAs. Target knockdowns for sense and antisense strands of siRNA EGFPS1A (a) and EGFPS1B (b) in 21-mer and Dicer substrate formats were carried out using co-transfections of the siRNAs with either the psi-EGFP-S sense reporter (red) or psi-EGFP-AS antisense reporter (black) in HEK293 cells. For both targets the 21-nt EGFPS1 siRNAs (left) or 25/27 DsiRNAs (right) were tested at various concentrations. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections). Sequences and IC50 values of each siRNA are shown. For the Dicer substrate siRNAs, the lower case letters represent deoxyribonucleotide containing bases which block Dicer entry.

Mentions: To discriminate the selective binding aspect of Dicer from direct incorporation of the siRNAs into Ago2, we modified EGFPS1A to generate an asymmetric 27/25-nt-long Dicer substrate form (DsiRNA)—a blunt end with two deoxyribonucleotide bases at the 3′-end of the passenger strand—forcing the duplex to be bound and undergo Dicer cleavage activity. EGFPS1B was also modified to a DsiRNA form for direct comparison, and the IC50 values were determined for both DsiRNAs (Figure 10a and b). For both EGFPS1A and B DsiRNAs, the strand harboring the 2-nt 3′ overhang was predominantly selected as the guide strand (IC50, Dsi-S1A-AS = 31.9 pM, IC50, Dsi-S1A-Sense = 101 pM; IC50, Dsi-S1B-AS = 27.5 pM, IC50, Dsi-S1B-Sense = 433 pM), consistent with previous observations (40). In contrast, the dominant selection of the antisense strand (relative to the sense EGFP target) was absent or less pronounced with the use of 19 + 2 siRNAs. Both the dsiRNAs and siRNAs form dicer-dependent complexes in whole-cell extracts (57) (Supplementary Figure S6). These results support a model in which the orientation of Dicer binding to its substrates sets the preference for which strand will be chosen to act as the guide strand. Taken together, our observations suggest that the initial Dicer–DsiRNA interaction is an important determinant for establishing siRNA-strand selection in RISC and ultimately the potency of the siRNAs. The Dicer substrates force Dicer to enter the RNA via its PAZ domain and the 2-base 3′ overhang of the Dicer substrate whereas 19 + 2 siRNAs allow Dicer to bind from either end using the 2-base 3′ overhang for PAZ domain interactions.Figure 10.


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)

Comparative analyses of guide strand selection for 21-mer and 25/27-mer Dicer substrate siRNAs. Target knockdowns for sense and antisense strands of siRNA EGFPS1A (a) and EGFPS1B (b) in 21-mer and Dicer substrate formats were carried out using co-transfections of the siRNAs with either the psi-EGFP-S sense reporter (red) or psi-EGFP-AS antisense reporter (black) in HEK293 cells. For both targets the 21-nt EGFPS1 siRNAs (left) or 25/27 DsiRNAs (right) were tested at various concentrations. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections). Sequences and IC50 values of each siRNA are shown. For the Dicer substrate siRNAs, the lower case letters represent deoxyribonucleotide containing bases which block Dicer entry.
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Figure 10: Comparative analyses of guide strand selection for 21-mer and 25/27-mer Dicer substrate siRNAs. Target knockdowns for sense and antisense strands of siRNA EGFPS1A (a) and EGFPS1B (b) in 21-mer and Dicer substrate formats were carried out using co-transfections of the siRNAs with either the psi-EGFP-S sense reporter (red) or psi-EGFP-AS antisense reporter (black) in HEK293 cells. For both targets the 21-nt EGFPS1 siRNAs (left) or 25/27 DsiRNAs (right) were tested at various concentrations. Target-specific Renilla luciferase expression was normalized to the control Firefly luciferase expression for all replicates (determined from multiple co-transfections). Sequences and IC50 values of each siRNA are shown. For the Dicer substrate siRNAs, the lower case letters represent deoxyribonucleotide containing bases which block Dicer entry.
Mentions: To discriminate the selective binding aspect of Dicer from direct incorporation of the siRNAs into Ago2, we modified EGFPS1A to generate an asymmetric 27/25-nt-long Dicer substrate form (DsiRNA)—a blunt end with two deoxyribonucleotide bases at the 3′-end of the passenger strand—forcing the duplex to be bound and undergo Dicer cleavage activity. EGFPS1B was also modified to a DsiRNA form for direct comparison, and the IC50 values were determined for both DsiRNAs (Figure 10a and b). For both EGFPS1A and B DsiRNAs, the strand harboring the 2-nt 3′ overhang was predominantly selected as the guide strand (IC50, Dsi-S1A-AS = 31.9 pM, IC50, Dsi-S1A-Sense = 101 pM; IC50, Dsi-S1B-AS = 27.5 pM, IC50, Dsi-S1B-Sense = 433 pM), consistent with previous observations (40). In contrast, the dominant selection of the antisense strand (relative to the sense EGFP target) was absent or less pronounced with the use of 19 + 2 siRNAs. Both the dsiRNAs and siRNAs form dicer-dependent complexes in whole-cell extracts (57) (Supplementary Figure S6). These results support a model in which the orientation of Dicer binding to its substrates sets the preference for which strand will be chosen to act as the guide strand. Taken together, our observations suggest that the initial Dicer–DsiRNA interaction is an important determinant for establishing siRNA-strand selection in RISC and ultimately the potency of the siRNAs. The Dicer substrates force Dicer to enter the RNA via its PAZ domain and the 2-base 3′ overhang of the Dicer substrate whereas 19 + 2 siRNAs allow Dicer to bind from either end using the 2-base 3′ overhang for PAZ domain interactions.Figure 10.

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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