title: In vitroReconstitution Assay of miRNA Biogenesis by Arabidopsis DCL1.
abstract: microRNAs (miRNAs) are small non-coding RNAs, regulating most if not all, biological processes in eukaryotic organisms. miRNAs are initially processed from primary transcripts (pri-miRNAs) to produce miRNA precursors (pre-miRNAs), that are further processed into miRNA and its complementary strands (miRNA/*). In Arabidopsis, and possibly other plants, the processing from pri-miRNAs to pre-miRNAs and from pre-miRNAs to miRNA/* are both implemented through Dicer-like 1 (DCL1) complexes. Recently, we demonstrated isolation of DCL1 complexes of unprecedented quality fromin planta.We further successfully reconstituted DCL1 cleavage assaysin vitrothat were able to fully recapitulatein vivomiRNA biogenesis. Here we provide a detailed protocol of DCL1 reconstitution assays. The protocol comprises three major parts (Figure 1): 1) Preparation of pri- and pre-miRNA transcripts (Procedures A-C); 2) Purification of the recombinant Arabidopsis DCL1 machinery fromNicotiana benthamiana(N. benthamiana) through immunoprecipitation (IP) (Procedures D and E); and 3)in vitroprocessing of radioisotope-labeled pri- or pre-miRNAs using the isolated DCL1 complexes (Procedure F). It is our desire that the protocol be a powerful tool for the RNAi community to study mechanistic issues or to develop RNA silencing technologies.
authors.affiliation: College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
authors.forename: Tian
authors.initials: T
authors.lastname: Wang
authors.affiliation: Department of Biochemistry and Biophysics, Institute of Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA.
authors.forename: Claudia
authors.initials: C
authors.lastname: Castillo-González
authors.affiliation: College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
authors.forename: Lan
authors.initials: L
authors.lastname: You
authors.affiliation: College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
authors.forename: Rui
authors.initials: R
authors.lastname: Li
authors.affiliation: College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
authors.forename: Liwei
authors.initials: L
authors.lastname: Wen
authors.affiliation: College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China.
authors.forename: Hongliang
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Department of Biochemistry and Biophysics, Institute of Plant Genomics and Biotechnology, Texas A&M University, College Station, TX, USA.
authors.forename: Xiuren
authors.initials: X
authors.lastname: Zhang