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 generated a protein-RNA tethering system which functions on an endogenous Arabidopsis RNA that is easily 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, deadenylase protein and translation factor to the RNA, which function to tag, decay and boost protein production, respectively. We demonstrated that our tethering system 1) is sufficient to engineer the downstream fate of an RNA, 2) enables the determination of any protein’s function upon recruitment to an RNA, and 3) can be used to discover new interactions with RNA-binding proteins. Examination of small RNA accumulation from the SOC1 mRNA and other regions in Arabidopsis thaliana lines with constructs that tether specific proteins to SOC1 and controls.