title: Pre-microRNA processing activity in nuclear extracts from Arabidopsis suspension cells.
abstract: MicroRNAs (miRNAs) play important roles in a variety of biological phenomena, such as development and responses to abiotic and biotic stresses, by regulating complementary target transcripts. Primary MIRNA transcripts (pri-miRNAs) are processed into miRNA/miRNA* duplexes via pre-miRNA intermediates by DICER family proteins. In plants miRNA/miRNA* duplexes are produced from pri-miRNAs exclusively by a complex containing DICER-LIKE1 (DCL1), Hyponastic leaves1 (HYL1) and SERREATE (SE) in the nucleus. Pri-miRNA or pre-miRNA processing activity has been detected using recombinant DCL1, HYL1 and SE proteins expressed in insect cells and immunopurified DCL1-HYL1-SE-containing complexes that transiently co-overexpressed in Nicotiana benthamiana. However, the processing of pre-miRNAs into miRNA/miRNA* using nuclear extracts has not been reported. Here I report the detection of pre-miRNA processing in nuclear extracts prepared from Arabidopsis suspension cells. In the nuclear extracts including DCL1, small RNAs that were 21 nucleotides in length were excised from a part of miRNA/miRNA* regions on pre-miRNAs. This system is potentially useful for in vitro analyses of pre-miRNA processing.
authors.affiliation: Division of Plant and Microbial Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 2-1-2 Kannondai Tsukuba, Ibaraki, 305-8602, Japan. myoshika@affrc.go.jp.
authors.forename: Manabu
authors.initials: M
authors.lastname: Yoshikawa
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins; C420966:HYL1 protein, Arabidopsis; D035683:MicroRNAs; D016601:RNA-Binding Proteins; C000606277:SE protein, Arabidopsis; C402383:DCL1 protein, Arabidopsis; D043244:Ribonuclease III
keywords: Arabidopsis thaliana; DICER-LIKE1; MicroRNA; Nuclear extract; Pre-miRNA