Abstract
The results of genetic studies in Arabidopsis indicate that three proteins, the RNase III DICER-Like1 (DCL1), the dsRNA-binding protein HYPONASTIC LEAVES1 (HYL1), and the C2H2 Zn-finger protein SERRATE (SE), are required for the accurate processing of microRNA (miRNA) precursors in the plant cell nucleus. To elucidate the biochemical mechanism of miRNA processing, we developed an in vitro miRNA processing assay using purified recombinant proteins. We find that DCL1 alone releases 21-nt short RNAs from dsRNA as well as synthetic miR167b precursor RNAs. However, correctly processed miRNAs constitute a minority of the cleavage products. We show that recombinant HYL1 and SE proteins accelerate the rate of DCL1-mediated cleavage of pre- and pri-miR167b substrates and promote accurate processing.
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