Optimization of Existing RNA Visualization Methods Reveals Novel Dendritic mRNA Dynamics.

Background: Spatial-temporal control of mRNA translation in dendrites is important for synaptic plasticity. In response to pre-synaptic stimuli, local mRNA translation can be rapidly triggered near stimulated synapses to supply the necessary proteins for synapse maturation or elimination, and 3' untranslated regions (UTRs) are responsible for proper localization of mRNAs in dendrites. Although FISH is a robust technique for analyzing RNA localization in fixed neurons, live-cell imaging of RNA dynamics remains challenging.

Methods: In this study, we optimized existing RNA visualization techniques (MS2-tagging and microinjection of fluorescently-labeled mRNAs) to observe novel behaviors of dendritic mRNAs.

Results: We found that the signal-to-noise ratio (SNR) of MS2-tagged mRNAs was greatly improved by maximizing the ratio of the MS2-RNA to MS2 coat protein-fluorescent protein (MCP-FP) constructs, as well as by the choice of promoter. Our observations also showed that directly fluorescently labeled mRNAs result in brighter granules compared to other methods. Importantly, we visualized the dynamic movement of co-labeled mRNA/protein complexes in dendrites and within dendritic spines. In addition, we observed the simultaneous movement of three distinct mRNAs within a single neuron. Surprisingly, we observed splitting of these complexes within dendritic spines.

Conclusions: Using highly optimized RNA-labeling methods for live-cell imaging, one can now visualize the dynamics of multiple RNA / protein complexes within the context of diverse cellular events. Newly observed RNA movements in dendrites and synapses may shed light on the complexities of spatio-temporal control of gene expression in neurons.