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

Kumi Sakurai; Mohammed Amarzguioui; Dong-Ho Kim; Jessica Alluin; Bret Heale; Min-sun Song; Anne Gatignol; Mark A Behlke; John J Rossi

doi:10.1093/nar/gkq846

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

Kumi Sakurai, Mohammed Amarzguioui, Dong-Ho Kim, Jessica Alluin, Bret Heale, Min-sun Song, Anne Gatignol, Mark A Behlke, John J Rossi

MRC Human Genetics Unit

Research output: Contribution to journal › Article › peer-review

Abstract / Description of output

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.

Original language	English
Pages (from-to)	1510-25
Number of pages	16
Journal	Nucleic Acids Research
Volume	39
Issue number	4
DOIs	https://doi.org/10.1093/nar/gkq846
Publication status	Published - Mar 2011

Keywords / Materials (for Non-textual outputs)

Argonaute Proteins
Cell Extracts
Eukaryotic Initiation Factor-2
HCT116 Cells
HEK293 Cells
Humans
RNA Interference
RNA, Small Interfering
RNA-Binding Proteins
RNA-Induced Silencing Complex
Ribonuclease III

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10.1093/nar/gkq846

A role for human Dicer in pre-RISC loading of siRNAs
© The Author(s) 2010. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Final published version, 8.89 MBLicence: Creative Commons: Attribution Non-Commercial (CC-BY-NC)

http://nar.oxfordjournals.org/content/39/4/1510

Cite this

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title = "A role for human Dicer in pre-RISC loading of siRNAs",

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.",

keywords = "Argonaute Proteins, Cell Extracts, Eukaryotic Initiation Factor-2, HCT116 Cells, HEK293 Cells, Humans, RNA Interference, RNA, Small Interfering, RNA-Binding Proteins, RNA-Induced Silencing Complex, Ribonuclease III",

author = "Kumi Sakurai and Mohammed Amarzguioui and Dong-Ho Kim and Jessica Alluin and Bret Heale and Min-sun Song and Anne Gatignol and Behlke, {Mark A} and Rossi, {John J}",

year = "2011",

month = mar,

doi = "10.1093/nar/gkq846",

language = "English",

volume = "39",

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AU - Sakurai, Kumi

AU - Amarzguioui, Mohammed

AU - Kim, Dong-Ho

AU - Alluin, Jessica

AU - Heale, Bret

AU - Song, Min-sun

AU - Gatignol, Anne

AU - Behlke, Mark A

AU - Rossi, John J

PY - 2011/3

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N2 - 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.

AB - 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.

KW - Argonaute Proteins

KW - Cell Extracts

KW - Eukaryotic Initiation Factor-2

KW - HCT116 Cells

KW - HEK293 Cells

KW - Humans

KW - RNA Interference

KW - RNA, Small Interfering

KW - RNA-Binding Proteins

KW - RNA-Induced Silencing Complex

KW - Ribonuclease III

U2 - 10.1093/nar/gkq846

DO - 10.1093/nar/gkq846

M3 - Article

C2 - 20972213

SN - 0305-1048

VL - 39

SP - 1510

EP - 1525

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 4

ER -

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

Abstract / Description of output

Keywords / Materials (for Non-textual outputs)

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Cite this