Cuerda-Gil Diego, Hung Yu-Hung, Panda Kaushik, Slotkin R Keith
Donald Danforth Plant Science Center, St. Louis, MO, USA.
Department of Molecular Genetics, The Ohio State University, Columbus, OH, USA.
Plant Methods. 2022 Jun 4;18(1):75. doi: 10.1186/s13007-022-00907-w.
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 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 target 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.
RNA转录本的分选决定了它们最终的转录后命运，如翻译、衰变或通过RNA干扰（RNAi）降解。RNA被分选为不同命运是由它们与RNA结合蛋白的相互作用介导的。虽然已经鉴定出数百种RNA结合蛋白，但将RNA分选到不同途径的具体机制大多仍不清楚。特别是在植物中，这是由于缺乏可靠的蛋白质-RNA人工拴系工具，而这些工具对于在体内确定蛋白质作用于RNA的机制是必需的。在此，我们构建了一个蛋白质-RNA拴系系统，该系统作用于一种内源性拟南芥RNA，通过定量开花时间表型对其进行追踪。与其他在植物中尝试过的蛋白质-RNA拴系系统不同，我们的系统避免了意外触发RNAi。我们成功地在体内将一个蛋白质表位、去腺苷酸化酶蛋白和翻译因子拴系到目标RNA上，它们分别起到标记、衰变和促进蛋白质产生的作用。我们证明，我们的拴系系统（1）足以设计RNA的下游命运，（2）能够确定任何蛋白质在被招募到RNA上时的功能，并且（3）可用于发现与RNA结合蛋白的新相互作用。
