Abstract
RNA-Protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA/protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-Protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for in vivo visualization of RNA-Protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) (Schonberger et al., 2012). Target RNA is tagged with 6xMS2 and MCP and RNA binding protein are fused with YFP fragments. DNA constructs encoding such fusion RNA and proteins are infiltrated into tobacco leaves with Agrobacterium suspensions. RNA-Protein interaction in vivo is observed by confocal microscope.
Keywords: Long non-coding RNA, RNA-Protein interaction, TriFC, Tobacco transient expression
Background
Recently, a variety of types of long-noncoding RNAs (lncRNAs) has been identified and shown to play important roles in transcriptional regulation and chromatin modification (St Laurent et al., 2015). So far, most of the molecular mechanisms for lncRNA-mediated functions are closely related with RNA-Protein interactions (St Laurent et al., 2015). Therefore, an experiment for RNA-Protein interaction is a key step in functional study of lncRNAs. In plants, molecular functions of lncRNAs are only beginning to be characterized, and the molecular basis of lncRNA-mediated gene regulation is still poorly understood. Though techniques for RNA visualization in plants have been well developed, visual assay for RNA-Protein interaction in plant is still poorly developed (Christensen et al., 2010). To develop the visual assay for RNA-Protein interaction in plants, we modified and combined MS2 system for RNA imaging technique with conventional BiFC system for protein-protein interaction (Schonberger et al., 2012) (Figure 1D). We generated binary Gateway vectors (pBA3130, 3132, 3134, and 3136) for transient BiFC assay (Seo et al., 2017) and got binary Gateway vectors (pBA-GW-6xMS2 and pBA-6xMS2-GW) for RNA tagging from Dr. Ulrich Z. Hammes (Schonberger et al., 2012). We tested and confirmed this TriFC assay was working well in plants with lncRNA, ELENA1, and MED19a protein (Seo et al., 2017). TriFC assay in plants will provide new insights in interaction between lncRNAs and proteins.
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Acknowledgments
We thank Dr. Ulrich Z. Hammes for the Gateway 6xMS2 tagging vectors. This work was supported by Singapore NRF RSSS Grant (NRF-RSSS-002).
References
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