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 from in planta. We further successfully reconstituted DCL1 cleavage assays in vitro that were able to fully recapitulate in vivo miRNA 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 from Nicotiana benthamiana (N. benthamiana) through immunoprecipitation (IP) (Procedures D and E); and 3) in vitro processing 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.
Keywords: Microprocessor, Pri-miRNA, MiRNA, Dicer, Arabidopsis
Materials and Reagents
Equipment
Procedure
Note: Please see Figure 1 for a schematic diagram of the six steps described below. Figure 1. A schematic diagram of procedure
Representative data
Representative data could refer to the Figures 2, 3, 4, 5, 6, 7 and 8 from Zhu et al. (2013), as well as supplementary Figures 2, 3, 4, 5, 6 and 9.
Notes
Recipes
Acknowledgments
This work was supported by grants from the US National Science Foundation (NSF) CAREER (MCB-1253369), the US National Institutes of Health (R21AI097570) and the Welch foundation (A-1777) to X.Z, and also supported by Chinese Universities Scientific Fund (2014RC006) to H.Z.
References
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