Development of fluorescent biosensors probing RNA function

Live cell imaging of RNA or their protein binding partners is crucial to obtain an authentic picture of RNA transcription, processing and trafficking. The development of methods for tracking of specific target RNAs in diverse cellular systems has been approached by different strategies including synthetic dyes, molecular beacons and genetically encoded RNA labels. In this work, we challenged current fluorescent protein RNA labeling methods by testing their performance for RNA visualization in neurons. Lack of dynamic behavior revealed the limitations of these current approaches and subsequently the need for novel on-off RNA reporters. Consequently, we designed synthetic and genetically encodable RNA biosensors with an RNA-dependent signal output and established tests suitable for analyzing their performance in vitro, in bacterial and eukaryotic systems. Application of synthetic on-off RNA dyes was hampered by impermeability or low specificity within the cellular environment. We engineered genetically encoded FRET RNA reporters based on the interaction of viral or synthetic RNA-peptide binding partners. Intramolecular FRET between two fluorescent proteins occurs only if the interspaced peptide undergoes a conformational change upon high-affine RNA aptamer binding. One of the identified sensors based on the Rsg1.2 peptide and the HIV Rev Responsive Element (RRE) RNA aptamer was called VAmPIRe (Viral Aptamer binding Peptide based Indicator for RNA detection). Its dynamic range was improved by engineering both the RNA aptamer tag and the fusion protein by random linker insertion and targeted mutagenesis. We demonstrate that the system is quantitative, reversible and can be used to tag RNAs of interest with high specificity in vitro and in living bacteria and mammalian cells. Besides tracking of specific RNAs, biosensors studying the function of RNA binding proteins like RNA helicases can provide further insights into RNA-based processes. Particularly interesting, the RNA helicase eIF4AIII is situated at the core of the Exon Junction Complex (EJC), a multi-protein complex involved in splicing, translation and RNA quality surveillance. We have designed the FRET-based fluorescent biosensor REFlex (Reporter of eIF4AIII Flexure) that monitors the conformational change of eIF4AIII underlying its transition from the open to the closed state. Generally, in vitro FRET monitoring using REFlex can be applied to reveal components that modulate EJC complex stability. More specifically, we have