Visualizing protein–RNA interactions inside cells by fluorescence resonance energy transfer

Approaches for studying protein–protein interactions in living cells have been broadly developed, but the temporal and spatial association of proteins with nucleic acids has been less explored. Here, we report a novel approach to study and visualize the association of an RNA-binding protein with its native RNA target in situ by fluorescence resonance energy transfer (FRET). The RNA-binding protein is tagged with a yellow variant of GFP and the RNA stained with SytoxOrange. RNA binding results in a decrease of the fluorescence lifetime of YFP due to FRET, which can be measured by fluorescence lifetime imaging microscopy (FLIM). With this method we analyzed the RNA binding of the alternative splicing repressors PTB and Raver1 and could show an RNA-specific FRET signal. Interestingly, PTB and Raver1 were bound to RNAs all over the nucleus, as expected, but additionally interacted with RNAs in the perinucleolar compartment (PNC), where only noncoding Pol III transcripts are present.