A plant tethering system for the functional study of protein-RNA interactions in vivo

The sorting of RNA transcripts dictates their ultimate post-transcriptional fates, such as translation, decay or degradation by RNA interference (RNAi). This sorting of RNAs into distinct fates is mediated by their interaction with RNA-binding proteins. While hundreds of RNA binding proteins have been identified, which act to sort RNAs into different pathways is largely unknown. Particularly in plants, this is due to the lack of reliable protein-RNA artificial tethering tools necessary to determine the mechanism of protein action on an RNA in vivo. Here we used minimal Arabidopsis BRN1 RNA binding domains to generate a protein-RNA tethering system which functions on an endogenous Arabidopsis RNA called SOC1 that is tracked by the quantitative flowering time phenotype. Unlike other protein-RNA tethering systems that have been attempted in plants, our system circumvents the inadvertent triggering of RNAi. We successfully in vivo tethered a protein epitope to the RNA and identified new proteins that were previously unknown to interact during BRN1-RNA binding. This protein-RNA tethering system enables the future determination of any protein’s function upon recruitment to an RNA and can be used to discover new interactions with RNA-binding proteins.