Uncovering mechanisms of axonal mRNA transport
Axonally synthesized proteins play important roles in neurodegeneration as well as neuropathic pain. At least two different mechanisms contribute to regulation of localized protein synthesis in neurons: mRNA transport/localization and localized mRNA translation. To localize individual mRNAs into neuronal processes, a mechanism must exist to enable selection of the mRNAs to be transported into dendrites and axons – in regenerating axons, both the rate and specificity of this transport can be regulated. Additionally, the translational efficiency of individual mRNAs can be regulated once the mRNAs reach the correct subcellular locale. My primary goal for the current research is to investigate the molecular mechanisms underlying axonal RNA localization, in particular, the specificity of axonal RNA transport and translation during neural repair and regeneration.
In addition to high-throughput methods exploiting the local transcriptome, we have been utilizing the single-molecule tracking along with real-time visualization of nascent protein synthesis. To this end, we have devoted substantial effort to develop methods to visualize axonal mRNAs by quantitative fluorescence in situ hybridization (qFISH), including localized mRNAs encoded by transfected reporter constructs carrying RNA localization elements (figures below).
Endogenous mRNAs localize into distal axons of sensory neurons. Confocal FISH-IF images of naïve vs. injured L4-5 DRGs and sciatic nerves in vivo (top) and DRG cultures (bottom) show increased signal for NMP35 (top, arrows) and amphoterin (bottom, arrowheads) mRNAs in distal axons whereas a decrease in the neuronal cell bodies. Scale bars, 3 μm for nerves (axons) and 10 μm for DRGs.
To identify the regions (cis-elements) within the localizing mRNAs needed for localization, I developed fluorescence recovery after photobleaching (FRAP) approach for biological reporter mRNA translation, where translation dependence of recovery can be directly tested (Figure below).
GAP43 3’UTR is sufficient for localization and translation of a reporter mRNA in axons. (left panels) Representative time-lapse images for FRAP analyses of DRG neurons transfected with the indicated constructs. The boxed regions represent regions of interests that were photobleached. Arrows indicate growth corns. Scale bars, 50 μm. (right panels) Quantifications of multiple time-lapse sequences in (left panels).
Coupling qFISH with FRAP analysis has provided a rigorous test for axonal RNA localization. We found that cis-elements within untranslated regions (UTRs) of the localizing mRNAs determine whether an mRNA is tagged for transport to subcellular regions and play central roles in regulating gene expression through translational efficiency and/or mRNA stabilization.