The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1.
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.
Dong
Zhicheng
Z
Biology Department and Huck Institutes of Life Sciences, Pennsylvania State University, University Park, PA 16802, USA.
Han
Meng-Hsuan
MH
Fedoroff
Nina
N
D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D007313:Insecta; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D000072099:Serrate-Jagged Proteins
D029681:Arabidopsis Proteins; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; C420966:HYL1 protein, Arabidopsis; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D035683:MicroRNAs; D018749:RNA, Plant; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C000606277:SE protein, Arabidopsis; D000072099:Serrate-Jagged Proteins; C402383:DCL1 protein, Arabidopsis; D043244:Ribonuclease III