title: A comprehensive analysis of precursor microRNA cleavage by human Dicer.
abstract: Dicer cleaves double-stranded RNAs (dsRNAs) or precursor microRNAs (pre-miRNAs) to yield ≈ 22-nt RNA duplexes. The pre-miRNA structure requirement for human Dicer activity is incompletely understood. By large-scale in vitro dicing assays and mutagenesis studies, we showed that human Dicer cleaves most, although not all, of the 161 tested human pre-miRNAs efficiently. The stable association of RNAs with Dicer, as examined by gel shift assays, appears important but is not sufficient for cleavage. Human Dicer tolerates remarkable structural variation in its pre-miRNA substrates, although the dsRNA feature in the stem region and the 2-nt 3'-overhang structure in a pre-miRNA contribute to its binding and cleavage by Dicer, and a large terminal loop further enhances pre-miRNA cleavage. Dicer binding protects the terminal loop from digestion by S1 nuclease, suggesting that Dicer interacts directly with the terminal loop region.
authors.affiliation: Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Feng
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Zhang
authors.forename: Paul
authors.initials: P
authors.lastname: Graves
authors.forename: Yan
authors.initials: Y
authors.lastname: Zeng
mesh_terms: D001483:Base Sequence; D053487:DEAD-box RNA Helicases / Q000737:chemistry*; D024202:Electrophoretic Mobility Shift Assay; D005656:Fungal Proteins / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D055029:Inverted Repeat Sequences; D007700:Kinetics; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D059367:RNA Cleavage; D012322:RNA Precursors / Q000737:chemistry*; D043244:Ribonuclease III / Q000737:chemistry*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D035683:MicroRNAs; D012322:RNA Precursors; C408087:DICER1 protein, human; D043244:Ribonuclease III; C526098:Endonuclease S1, Aspergillus; D015719:Single-Strand Specific DNA and RNA Endonucleases; D053487:DEAD-box RNA Helicases