1. To investigate the real-time dynamics of gene expression in the neural networks.
Using our in vivo RNA labeling technology (Park et al., Science, 343, 422, (2014); Figure 1), we are investigating the dynamics of gene expression that are involved in synaptic plasticity. The immediate early gene Arc is one of the crucial genes in regulating long-term memory formation. Arc has important functions in regulation of actin cytoskeletal dynamics, regulation of AMPA receptor endocytosis, and homeostatic plasticity. Studies indicate that the expression of Arc is tightly coupled to synaptic plasticity in neuronal circuits in vivo.
Using the mouse models that we generated, we are investigating the dynamic regulation of β-actin and Arc mRNA in live mouse brain. By monitoring transcription or localization of mRNA simultaneously with calcium imaging, we study how neuronal activity patterns are transduced into the changes in gene expression and structural plasticity. We also develop
quantitative analysis algorithms to investigate the spatial and temporal regulation of single mRNA and its physiological impact within the native tissue environment. Our long-term vision is to extend this in vivo RNA imaging technique to other protein-coding genes as well as recently discovered non-coding RNA or extracellular RNA. These approaches will allow us to determine the regulation of a specific RNA in live neural network with unprecedented spatial and temporal resolution.
2. To study activity-dependent mRNP interactomes in neurons.
From transcription to decay, the fate of mRNA is orchestrated by many RNA-binding proteins (RBPs) in dynamically regulated ribonucleoprotein (RNP) complex. There have been increasing interests in identifying the RBP repertoire since many neurodegenerative diseases have been linked to defects in RBPs. We are interested in understanding how these RBPs are interacting with a specific target mRNA in neurons in vivo.
Arc mRNA is induced by synaptic activity, transported to the activated dendrites, translated locally, and degraded by the nonsense-mediated decay (NMD) pathway. Since Arc mRNA has a short half-life of 47 minutes, newly synthesized mRNA can be followed from transcription to decay with a very low background of pre-existing mRNA. We utilize single molecule imaging in live neurons to investigate the interaction of RBPs with Arc mRNA at different stages of the RNA lifecycle. These experiments will provide a major step forward in systems-level studies of mRNP complex in learning and memory, and in neurological diseases.
3. To develop non-invasive imaging of transcription in live animals.
By using high-resolution optical imaging techniques, we can routinely detect single molecules of mRNA labeled with many GFPs. The next step is to develop non-invasive techniques to visualize RNA in living animals. The main challenge in applying the MS2-GFP system for whole animal imaging is the background fluorescence of MCP-GFP present in the cells regardless of the expression of MBS-tagged mRNA. When imaging a whole animal, it is difficult to achieve sub-cellular resolution; a new strategy will be necessary to report RNA expression with a minimal background.
We employ split protein complementation approaches to minimize the background. There are various two-hybrid systems for in vivo imaging modalities such as bioluminescence, magnetic resonance imaging (MRI), positron emission tomography (PET), and near IR fluorescence. These newly developed protein-protein interaction reporters for non-invasive imaging could be applied to RNA imaging in live animals. Moreover we could use this technique in conjunction with various disease mouse models to study the pathologies associated with abnormal gene expression. This novel technology will provide a foundation for better understanding of the disease mechanisms and the development of new therapies.