Substrate-Specific Kinetics of Dicer-Catalyzed RNA Processing

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-miRNA substrate compared to a pre-siRNA substrate under both single and multiple turnover conditions. Maximal cleavage rates (Vmax) 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.

Research Highlights
► Human Dicer shows significant variability in dicing efficiency in a substrate sequence- and/or structure-dependent manner.
► We report a clear preference for pre-miRNAs over pre-siRNAs.
► The Dicer-associated dsRNA binding protein TRBP stimulates a ∼ 5-fold increase in endonucleolytic activity with both kinds of substrates.
► This stimulatory effect of TRBP is most likely dependent on the high RNA binding affinity attributable to dsRBD1 and dsRBD2.