The development and maintenance of neurons and synaptic connections are highly complex processes, in part due to the massive cytoplasmic volume and complex branching morphology of axons and dendrites. As one mechanism, it is well appreciated that RNA localization and local translation are required to precisely regulate protein homeostasis at synapses. Indeed, loss of FMRP in Fragile X Syndrome, or other impairments to RNA localization and local translation at synapses, likely contribute to brain disorders. To better understand RNA localization and local translation in neurons, we must elucidate the RNA cis-elements, RBP trans-factors, and cytoskeletal motors mediating these processes. Although ongoing efforts have demonstrated how RNA binding proteins (RBPs) can regulate local translation at post-synaptic sites, there still exists a major gap in our understanding of how RBPs transport RNAs to regulate synaptic function. Fortunately, recent observations provide clues about fruitful lines of investigation. For example, multiple studies report that distally localized RNAs are enriched for cis-elements targeted by Muscleblind-like (MBNL) proteins. Although these observations suggest that MBNL may be a major player in localizing RNAs to the pre- and post-synapse, we still lack a mechanistic understanding for how MBNL proteins may achieve this task, or what functions depend on MBNL-mediated RNA localization. This line of research has important implications for the neurological disease myotonic dystrophy (dystrophia myotonica, DM), in which MBNLs are depleted by toxic CUG repeats. Therefore, an emerging hypothesis is that RNA localization functions of MBNL are important for proper synapse function, and that mis-localized RNAs might account for some neurological features of DM patients, particularly early in disease. Here, using MBNL depletion and DM-associated models, we propose to identify specific functions for the localization of MBNL targets.
Aim 1 will elucidate mechanisms of MBNL-mediated mRNA localization in neurons. We will define the RNA targets that are localized by MBNL in the pre- and post-synapse. We will characterize dynamic properties of motile MBNL RNA granules in live neurons and identify cytoskeletal motors and adaptors associated with these granules. Using genomics, live cell imaging, and biochemical approaches, we will establish mechanisms of how MBNL-interacting RNAs are transported.
Aim 2 will define functions conferred by MBNL-dependent RNA localization using models of synapse development and function, and models of myotonic dystrophy. By depleting cytoplasmic MBNL and other proteins required for MBNL-dependent RNA localization, we will assess cellular functions dependent on this process. We will identify specific neuronal functions, such as synaptic vesicle release, that depend on proper localization of mRNAs by MBNL proteins. The impact of this research is to better understand how RNA localization and local translation confers important synaptic functions and how they may go awry in DM. As few RNA binding proteins have been linked to motors, this may evolve into a unifying model for mRNA transport to synapses.
The proposed basic research is focused on better understanding how RNAs are localized and translated in neurons. Perturbations to these processes occur in many neurological diseases, but we do not have a good understanding of how RNA localization occurs. This research has implications for many neurological diseases.