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Published: Jul 5, 2018 DOI: 10.21769/BioProtoc.2909 Views: 6896
Edited by: Amey Redkar Reviewed by: Marisa Rosa
Abstract
RNA immunoprecipitation (RIP) is an antibody-based technique used to map in vivo RNA-protein interactions. DBR1, an RNA debranching enzyme, is responsible for the debranching of lariat RNA, for the degradation and turnover of lariat RNAs. It is well known that primary miRNA (Pri-miRNA) is recognized and further processed into mature miRNA by the Dicing complex mainly composed of DCL1 and HYL1. Due to the low abundance of pri-miRNAs, RIP followed qRT-PCR has been widely used to evaluate the binding efficiency of the Dicing complex with pri-miRNAs in previous studies. Therefore, the genome-wide evaluation of the Dicing complex with pri-miRNAs is lacking. With the improvement of high-throughput sequencing technologies, we successfully used RIP-seq to compare the binding efficiency of the Dicing complex with pri-miRNAs between wild-type and the dbr1-2 mutant in our recent study. In this protocol, we provide a detailed description of RIP-seq using GFP-trap beads in HYL1-YFP and DCL1-YFP transgenic plants between two different genotypes. This method can be used to assess the binding of pri-miRNAs with the Dicing complex in Arabidopsis, and it can be applied to other RNA binding proteins in plants.
Keywords: DBR1Materials and Reagents
Equipment
Procedure
Category
Plant Science > Plant molecular biology > RNA
Molecular Biology > RNA > RNA-protein interaction
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