Substrate-Specific Kinetics of Dicer-Catalyzed RNA Processing

JOURNAL OF MOLECULAR BIOLOGY

卷:404

Article

Chakravarthy, Srinivas^1,2  Sternberg, Samuel H.³  Kellenberger, Colleen A.³  Doudna, Jennifer A.^1,2,3,4 

[1] Univ Calif Berkeley, Howard Hughes Med Inst, Berkeley, CA 94720 USA

[2] Univ Calif Berkeley, Dept Mol & Cell Biol, Berkeley, CA 94720 USA

[3] Univ Calif Berkeley, Dept Chem, Berkeley, CA 94720 USA

[4] Univ Calif Berkeley, Lawrence Berkeley Lab, Phys Biosci Div, Berkeley, CA 94720 USA

关键词: Dicer;    siRNA;    miRNA;    TRBP;    kinetics;   

DOI  :  10.1016/j.jmb.2010.09.030

来源: Elsevier

【 摘 要 】

The specialized ribonuclease Dicer plays a central role in eukaryotic gene expression by producing small regulatory RNAs microRNAs (miRNAs) and short interfering RNAs (siRNAs) from larger double-stranded RNA (dsRNA) substrates. Although Dicer will cleave both imperfectly base-paired hairpin structures (pre-miRNAs) and perfect duplexes (pre-siRNAs) in vitro, it has not been clear whether these are mechanistically equivalent substrates and how dsRNA binding proteins such as trans-activation response (TAR) RNA binding protein (TRBP) influence substrate selection and RNA processing efficiency. We show here that human Dicer is much faster at processing a pre-rniRNA substrate compared to a pre-siRNA substrate under both single and multiple turnover conditions. Maximal cleavage rates (V-max) calculated by Michaelis-Menten analysis differed by more than 100-fold under multiple turnover conditions. TRBP was found to enhance dicing of both substrates to similar extents, and this stimulation required the two N-terminal dsRNA binding domains of TRBP. These results demonstrate that multiple factors influence dicing kinetics. While TRBP stimulates dicing by enhancing the stability of Dicer substrate complexes, Dicer itself generates product RNAs at rates determined at least in part by the structural properties of the substrate. (c) 2010 Elsevier Ltd. All rights reserved.

【 授权许可】

Free