Imaging spatiotemporal mRNA dynamics at subcellular resolution with a bright fluorogenic RNA

[Speaker] Tetsuro Ariyoshi:1
[Co-author] Yasushi Okada:1
1:Laboratory for Cell Polarity Regulation, RIKEN BDR, Japan

Recent advances in single-cell analysis techniques and microscopy enabled the monitoring of the transcriptional activity at single-cell resolution. Rapidly maturating and fast degrading fluorescent or luminescent proteins has been widely used for monitoring promoter activity so far, but the signal from these proteins do not reflect the transcriptional activity directly. Combinations of fluorescent protein-tagged RNA-binding proteins and RNA stem-loops, such as MS2 or PP7 stem-loop, have been developed for the direct visualization of RNA in living cells, however, this approach often suffers from the background signals of unbound fluorescent proteins which makes it difficult to precisely measure the expression levels of the mRNA of interest. Fluorogenic RNAs would be ideal for measuring the transcriptional activity, because their fluorescence signal directly reflects the amount of the target RNA. Spinach, Broccoli and their derivatives have been developed as fluorogenic RNAs that emit green fluorescence upon binding to a small molecule 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). However, their application was limited, mainly because their fluorescence signal was too weak to detect mRNA in living mammalian cells. Here we report a new fluorogenic RNA probe Romanesco, an improved version of Broccoli. The improved brightness of Romanesco enabled us to measure transcription activity in living mammalian cells in culture. Reporter constructs that contains a coding sequence of iRFP followed by Romanesco at the 3' end successfully reported the rise and fall of green fluorescent signal from Romanesco reflecting the promoter activity, while the iRFP signal was delayed. The linearity of Romanesco signal was verified by single-cell RT-qPCR analysis, but the iRFP signal intensity was not proportional to the Romanesco signal, which would demonstrate the cell-to-cell variance in the translational activity. By utilizing Romanesco-based fluorescent reporter system we have also revealed a large cell-to-cell heterogeneity in stimulus-triggered transcriptional dynamics in immune cells. Furthermore, the subcellular localization of the fluorescent signal visualized the RNA translocation from nucleoplasm to cytoplasm as well as the accumulation of newly synthesized RNA molecules around the nuclear speckles. Thus, Romanesco would serve as a versatile visualization probe for the transcription, dynamics and localization of RNA in living cells.
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