Howard Hughes Medical Institute, Berkeley, CA 94720, USA.
J Mol Biol. 2010 Dec 3;404(3):392-402. doi: 10.1016/j.jmb.2010.09.030. Epub 2010 Oct 13.
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 (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.
专门的核糖核酸酶 Dicer 通过从较大的双链 RNA（dsRNA）底物中产生小的调控 RNA- microRNAs（miRNAs）和短干扰 RNA（siRNAs），在真核生物基因表达中发挥核心作用。尽管 Dicer 在体外可以切割不完全碱基配对的发夹结构（pre-miRNAs）和完美的双链体（pre-siRNAs），但尚不清楚这些是否是具有相同机制的底物，以及 dsRNA 结合蛋白（如转激活反应（TAR）RNA 结合蛋白（TRBP））如何影响底物选择和 RNA 加工效率。我们在这里表明，在单轮和多轮条件下，与 pre-siRNA 底物相比，人 Dicer 处理 pre-miRNA 底物的速度要快得多。米氏分析计算的最大切割速率（V（max））在多轮条件下差异超过 100 倍。发现 TRBP 可以相似程度地增强两种底物的切割，这种刺激需要 TRBP 的两个 N 端 dsRNA 结合结构域。这些结果表明，多种因素影响切割动力学。虽然 TRBP 通过增强 Dicer-底物复合物的稳定性来刺激切割，但 Dicer 本身以至少部分由底物结构特性决定的速率产生产物 RNA。
