Curated Optogenetic Publication Database

Abstract: RNA-binding proteins (RBPs) play an essential role in regulating the function of RNAs in a cellular context, but our ability to control RBP activity in time and space is limited. Here, we describe the engineering of LicV, a photoswitchable RBP that binds to a specific RNA sequence in response to blue light irradiation. When fused to various RNA effectors, LicV allows for optogenetic control of RNA localization, splicing, translation and stability in cell culture. Furthermore, LicV-assisted CRISPR-Cas systems allow for efficient and tunable photoswitchable regulation of transcription and genomic locus labeling. These data demonstrate that the photoswitchable RBP LicV can serve as a programmable scaffold for the spatiotemporal control of synthetic RNA effectors.

Abstract: Fluorescent RNAs (FRs), aptamers that bind and activate fluorescent dyes, have been used to image abundant cellular RNA species. However, limitations such as low brightness and limited availability of dye/aptamer combinations with different spectral characteristics have limited use of these tools in live mammalian cells and in vivo. Here, we develop Peppers, a series of monomeric, bright and stable FRs with a broad range of emission maxima spanning from cyan to red. Peppers allow simple and robust imaging of diverse RNA species in live cells with minimal perturbation of the target RNA's transcription, localization and translation. Quantification of the levels of proteins and their messenger RNAs in single cells suggests that translation is governed by normal enzyme kinetics but with marked heterogeneity. We further show that Peppers can be used for imaging genomic loci with CRISPR display, for real-time tracking of protein-RNA tethering, and for super-resolution imaging. We believe these FRs will be useful tools for live imaging of cellular RNAs.