Mechanism of mRNA Localization and Localized Translation in Neurons

ABSTRACT The neuron is the basic cellular unit of the brain. For neurons to work properly, they must be plastic and constantly capable of changing in response to stimuli, forming and stabilizing new connections. This process requires proteins to be added to the new synaptic contact, and this in turn results from the targeting of mRNA to these sites of activity, as we have shown in our previous work. This is the molecular basis of learning and memory since the synapse is stabilized by the production of proteins in response to stimulation that is important for its function and structural integrity. How this mRNA is regulated in neurons to make the right protein at the right place and time has been the subject of our investigations over the years of this funding. This proposal exploits the tools we developed during the last funding period to address how mRNA is regulated in dendrites. We have expended considerable effort in the generation of genetically engineered mice wherein the loci of three neuronally expressed genes important for learning and memory have been tagged with stem loops that, when expressed in the mRNA bind to fluorescent proteins. The single mRNAs expressed from these genes can be imaged in living cells and extended into live tissues. We have taken care to verify that the tagging is neutral: it does not alter the behavior or affect memory formation in the mice. One of these tags is for Arc, an immediate early gene in response to neuronal stimulation important for consolidating long term memory. Unlike the constitutive -actin mRNA, which we showed sits at the place where it was last stimulated for hours, Arc mRNA localizes only for a few minutes, and degrades soon after. The current proposal reports on the progress to solving how transiently localized mRNAs can impact long term changes at the stimulated spines. The surprise was that Arc undergoes cycles of localization and translation in response to a single stimulus. Even more incredible is that the translation occurs spatially at the same spot, so the mRNA in the next cycle finds the site of previous localization and builds up a continuous “hotspot” of localized protein synthesis. This in contrast to the β-actin mRNA, which persists at the stimulated site, awaiting the next signal, wherein it will initiate another round of proteins. Because β-actin is a structural protein, the synaptic contact is built up with rounds of translation, consistent with a learning and memory paradigm that relies on repetitive stimulation. The current proposal is directed towards understanding the kinetics of translation hotspots, and their spatial overlap for different mRNAs with distinct roles in plasticity. We crossed the β-actin and Arc mice to homozygosity where both mRNAs were individually detectable by different colored fluorochromes in the same neuron. We have learned from this mouse that the two mRNAs were handled differently by the neuron, and traveled in independent “granules”, likely resulting from differences in their associated proteins. For instance, β-actin mRNA bound the zipcode binding protein, ZBP1 (IMP1) whereas Arc mRNA instead bound the protein FMRP. Further progress will elucidate the protein composition of each granule in more detail. We have made two more mice during the last funding period: a GCN4 epitope tagged (“Suntag”) Arc mouse that will allow us to see the translation sites of Arc protein using a fluorescent single chain antibody in living cells and tissues (we developed this tag previously), and a CaMKII mouse where the mRNA is distinguishable from either β-actin mRNA or Arc mRNA by hybrid fluorescent tags. This allows us now to contrast how the neuron handles each mRNA, for example in its localization and translation. Over the last funding period, an unexpected result was that the CaMKII mRNA localized in the spines, unlike either Arc or β-actin mRNA, that stayed at the base of the spines. This indicated that subtleties in the localization of these mRNAs may underlie a physiological purpose. We intend to investigate this by determining the sequences that likely direct this mRNA into the spines. Ultimately, we intend to find the proteins bound specifically to these mRNAs and how they might affect the regulation of their respective mRNAs. This will use the technologies of RNA editing and proximity labeling, which will allow us to interrogate the associated RNAs and proteins that make each species of mRNA granule unique.