Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP

Dicer is a multi‐domain RNase III‐related endonuclease responsible for processing double‐stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the pu...

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Bibliographic Details
Published in:The EMBO journal 2002-11, Vol.21 (21), p.5875-5885
Main Authors: Zhang, Haidi, Kolb, Fabrice A., Brondani, Vincent, Billy, Eric, Filipowicz, Witold
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
ATP
Base Sequence
DNA Primers
EMBO36
Endoribonucleases - isolation & purification
Endoribonucleases - metabolism
gene silencing
HeLa Cells
Humans
Hydrolysis
Mammals
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Ribonuclease III
ribonucleases
RNA Processing, Post-Transcriptional
RNA, Double-Stranded - chemistry
RNA, Double-Stranded - metabolism
RNA, Small Interfering - metabolism
RNAi
RNase III
siRNA
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Summary:Dicer is a multi‐domain RNase III‐related endonuclease responsible for processing double‐stranded RNA (dsRNA) to small interfering RNAs (siRNAs) during a process of RNA interference (RNAi). It also catalyses excision of the regulatory microRNAs from their precursors. In this work, we describe the purification and properties of a recombinant human Dicer. The protein cleaves dsRNAs into ∼22 nucleotide siRNAs. Accumulation of processing intermediates of discrete sizes, and experiments performed with substrates containing modified ends, indicate that Dicer preferentially cleaves dsRNAs at their termini. Binding of the enzyme to the substrate can be uncoupled from the cleavage step by omitting Mg 2+ or performing the reaction at 4°C. Activity of the recombinant Dicer, and of the endogenous protein present in mammalian cell extracts, is stimulated by limited proteolysis, and the proteolysed enzyme becomes active at 4°C. Cleavage of dsRNA by purifed Dicer and the endogenous enzyme is ATP independent. Additional experiments suggest that if ATP participates in the Dicer reaction in mammalian cells, it might be involved in product release needed for the multiple turnover of the enzyme.
ISSN:0261-4189
1460-2075
1460-2075
DOI:10.1093/emboj/cdf582