Live imaging of RNA and RNA splicing in mammalian cells via the dcas13a-SunTag-BiFC system.

Dynamic tracking of the localization of RNA molecules (nucleus and/or cytoplasm) and RNA splicing in living cells plays an important role in understanding their functions. However, a lack of dynamic imaging and high background fluorescence have been reported in the fluorescence in situ hybridization (FISH). Here, we developed a new tool, the dcas13a-SunTag-BiFC system, which fused the dLwacas13a and SunTag systems. dLwacas13a is used as a tracker to target specific RNAs, while SunTag recruits split Venus fluorescent proteins to label targeted RNAs. Our results showed that 4 × NLS-dCas13a-24 × SunTag-BiFC and 2 × NLS- dCas13a-24 × SunTag-BiFC systems can be used for imaging of endogenous RNA foci in the nucleus (Xist) and cytoplasm (Ppib and stress granules) in living cells, respectively. Compared to 12x MS2-MCP system, the dcas13a-SunTag-BiFC system showed a better performance of mRNA foci tracking in live cells. Furthermore, we confirmed the premature termination codon (PTC)-induced exon skipping of Oxt RNA using the dcas13a-SunTag-BiFC and MS2-MCP systems in the nucleus. Thus, the dcas13a-SunTag-BiFC system will facilitate the study of RNA localization in living cells and provide new insights into RNA translocation and splicing.