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In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6   

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Original research article

A brief version of this protocol appeared in:
Nature Plants
Mar 2017

Abstract

RNA-dependent RNA polymerases (RdRPs) in eukaryotes convert single-stranded RNAs into double-stranded RNAs, thereby amplifying small interfering RNAs that play crucial roles in the regulation of development, maintenance of genome integrity and antiviral immunity. Here, we describe a method of in vitro RdRP assay using recombinant Arabidopsis RDR6 prepared by an insect expression system. By using this classical biochemical assay, we revealed that RDR6 has a strong template preference for RNAs lacking a poly(A) tail. This simple method will be applicable to other RdRPs in Arabidopsis and different organisms.

Keywords: RNA-dependent RNA polymerase, Double-stranded RNA, RNA silencing, Small RNA, siRNA, RNA-DEPENDENT RNA POLYMERASE6, Arabidopsis thaliana

Background

RNA-dependent RNA polymerase (RdRP) genes have been found in all eukaryotic kingdoms–plants, fungi, protista and animals (Zong et al., 2009). They convert single-stranded RNAs (ssRNAs) into double-stranded RNAs (dsRNAs), thereby amplifying small interfering RNAs (siRNAs) that play crucial roles in various biological processes including regulation of development (Peragine et al., 2004; Li et al., 2005), maintenance of genome integrity (Volpe et al., 2002; Xie et al., 2004) and antiviral immunity (Mourrain et al., 2000; Yu et al., 2003; Garcia-Ruiz et al., 2010; Wang et al., 2010). In addition to this RdRP activity, RdRPs possess another enzymatic activity called terminal nucleotide transferase (TNTase) activity (Curaba and Chen, 2008; Aalto et al., 2010), which adds one or more nucleotides to the 3’ end of ssRNAs or dsRNAs in a template-independent manner.

Here, we describe a method for in vitro RdRP assay using recombinant Arabidopsis thaliana RDR6 prepared by an insect expression system. To accurately discriminate RdRP products from TNTase products, we designed two strategies: 1) elimination of single-stranded TNTase products by treating the reaction mixture with ssRNA-specific RNase I, and 2) electrophoresis of the reaction mixture on a native acrylamide gel, in which double-stranded RdRP products can be distinguished from single-stranded TNTase products based on their different mobility. By using this classical biochemical assay, we revealed that RDR6 has a strong template preference for the RNAs lacking a poly(A) tail (Baeg et al., 2017). This simple method should also be applied to other RdRPs in Arabidopsis and different organisms.

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Baeg, K., Tomari, Y. and Iwakawa, H. (2018). In vitro RNA-dependent RNA Polymerase Assay Using Arabidopsis RDR6. Bio-protocol 8(1): e2673. DOI: 10.21769/BioProtoc.2673.
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