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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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Analysis of proteins comprising the complex. Super shift assays. α-Dicer, α-TRBP, α-Ago2 or α-Ago1 antibodies were added to the HEK293 cell extract and incubated for 15 min prior to addition of either EGFPS1A or hnRNPH1 siRNA. Western analyses of proteins detected with the antibodies were also shown on the right. Of total protein, 50 µg for detection of Dicer and Ago1 or 150 µg for Ago2, TRBP and β-actin was loaded on 8% SDS–PAGE gels.
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Figure 6: Analysis of proteins comprising the complex. Super shift assays. α-Dicer, α-TRBP, α-Ago2 or α-Ago1 antibodies were added to the HEK293 cell extract and incubated for 15 min prior to addition of either EGFPS1A or hnRNPH1 siRNA. Western analyses of proteins detected with the antibodies were also shown on the right. Of total protein, 50 µg for detection of Dicer and Ago1 or 150 µg for Ago2, TRBP and β-actin was loaded on 8% SDS–PAGE gels.

Mentions: The results described above were carried out using either the HEK293 or HCT116 cell lines. In order to further explore the complex composition, we carried out additional biochemical experiments in the HEK293 cell extracts. The human RLC consists of Ago2, Dicer and TRBP (26–29). To determine whether any of these components are involved in the siRNA-complex binding observed in this study, we carried out antibody-mediated super-shift assays with HEK293 cell extracts using antibodies against Dicer, TRBP, Ago2 (and Ago1) to monitor complex formation with the EGFPS1A and hnRNPH1 siRNAs (Figure 6). Addition of the anti-Dicer antibody resulted in a strong super-shift of the complex whereas the anti-TRBP antibody yielded a weak but reproducible supershift. These results indicate that the complex observed in Figures 1–3 contain at least Dicer and TRBP, consistent with the previous observation by Pellino et al. (55). Although no supershifts were observed with the anti-Ago2 or anti-Ago1 antibodies, these antibodies did recognize the cognate proteins in the HEK293 whole cell extract (Figure 6).Figure 6.


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)

Analysis of proteins comprising the complex. Super shift assays. α-Dicer, α-TRBP, α-Ago2 or α-Ago1 antibodies were added to the HEK293 cell extract and incubated for 15 min prior to addition of either EGFPS1A or hnRNPH1 siRNA. Western analyses of proteins detected with the antibodies were also shown on the right. Of total protein, 50 µg for detection of Dicer and Ago1 or 150 µg for Ago2, TRBP and β-actin was loaded on 8% SDS–PAGE gels.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: Analysis of proteins comprising the complex. Super shift assays. α-Dicer, α-TRBP, α-Ago2 or α-Ago1 antibodies were added to the HEK293 cell extract and incubated for 15 min prior to addition of either EGFPS1A or hnRNPH1 siRNA. Western analyses of proteins detected with the antibodies were also shown on the right. Of total protein, 50 µg for detection of Dicer and Ago1 or 150 µg for Ago2, TRBP and β-actin was loaded on 8% SDS–PAGE gels.
Mentions: The results described above were carried out using either the HEK293 or HCT116 cell lines. In order to further explore the complex composition, we carried out additional biochemical experiments in the HEK293 cell extracts. The human RLC consists of Ago2, Dicer and TRBP (26–29). To determine whether any of these components are involved in the siRNA-complex binding observed in this study, we carried out antibody-mediated super-shift assays with HEK293 cell extracts using antibodies against Dicer, TRBP, Ago2 (and Ago1) to monitor complex formation with the EGFPS1A and hnRNPH1 siRNAs (Figure 6). Addition of the anti-Dicer antibody resulted in a strong super-shift of the complex whereas the anti-TRBP antibody yielded a weak but reproducible supershift. These results indicate that the complex observed in Figures 1–3 contain at least Dicer and TRBP, consistent with the previous observation by Pellino et al. (55). Although no supershifts were observed with the anti-Ago2 or anti-Ago1 antibodies, these antibodies did recognize the cognate proteins in the HEK293 whole cell extract (Figure 6).Figure 6.

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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