Structure-Function Studies of Translational Regulatory lncRNA BC200 and the Development of Synthetic RNA Imaging Probes

Abstract

Long noncoding RNAs (lncRNAs) have emerged as critical regulators of synaptic plasticity, in part through their ability to enact prompt changes to protein production far from the neuron’s nucleus. The lncRNA BC200 has been previously shown to modulate local translation in dendrites, although its mechanism is unknown. Using in vitro lysates, we show BC200 enhances noncanonical translation (i.e., from transcripts containing uORFs and IRESs) while simultaneously repressing cap-dependent translation. We also demonstrate that BC200 binds the synaptic plasticity-associated secondary messenger cyclic GMP as well as its inactive form GMP, the former of which further enhances BC200’s effect on noncanonical translation. Thus, we posit that BC200 potentially acts as a cGMP/GMP-responsive RNA sensor in neuronal dendrites, a novel phenomenon for a human long noncoding RNA. Additionally, we find that BC200 associates with proteins in the eIF2 regulatory network, suggesting a potential mechanism for BC200’s translational regulation. Taken together, our studies suggest that BC200 may function as a highly tunable molecular processor that can respond to environmental cues to regulate local translation in neurons. In conceptualizing future in cellulo assays of BC200 selective translation, we noted the scarcity of available RNA tracking technology for live cell microscopy. Additionally, although antibody derivatives, such as Fabs and scFvs, have revolutionized cellular imaging, quantification, and tracking of proteins, analogous tools and strategies are unavailable for cellular RNA visualization. Here, we developed synthetic anti-RNA scFv (sarabody) probes and their green fluorescent protein (GFP) fusions, and we demonstrated their potential to visualize RNA in live mammalian cells. This imaging strategy is analogous to the existing MCP–MS2 system for RNA visualization, but our approach additionally provides robust flexibility for developing target RNA-specific imaging modules, as epitope-specific probes can be selected from a library generated by diversifying the sarabody complementarity-determining regions. These results demonstrate that these first-of-their-kind immunofluorescent probes have tremendous potential for tracking mature RNAs and may aid in visualizing, quantifying, and examining the spatiotemporal dynamics of various RNAs and many cellular processes, such as BC200 selective translation regulation.