Visualizing single mRNAs and their topology at synapses

Certain neuronal mRNAs are transported to dendrites, where their local translation contributes to synaptic plasticity and learning and memory. Defects in RNA localization can have severe consequences for the function of cells and its association with several human diseases including fragile X mental retardation, spinal muscular atrophy and myotonic dystrophy. Transport of RNAs to dendrites and synapses is thought to occur in ribonucleoprotein particles (RNPs), however, little is known about the underlying components and their function in mRNA localization. We propose to apply a very sensitive in situ hybridization method, high-resolution imaging and sophisticated image analysis developed by our collaboration partners in order to detect single RNA molecules at individual synapses and to determine their topology and correlate it to their translational status. This study will allow us to gain unprecedented insight into when and where a particular mRNA is silent and when it becomes translated. We expect that this thorough analysis will greatly advance the mechanistic understanding of dendritic RNA localization and its contribution to learning and memory and disease.

Neuronal mRNAs are transported to dendrites, where their local translation contributes to synaptic plasticity, learning and memory formation. RNA-binding proteins (RBPs), e.g. the double-stranded RBP Staufen2, mediate the transport of these mRNAs to dendrites and synapses in the form of ribonucleoprotein particles (RNPs). In order to gain insight into the underlying components and their function in mRNA localization, the Kiebler lab has now pioneered the biochemical and functional characterization of Stau2-containing RNPs both at the protein and RNA level. We next investigated two high confidence Stau2 target mRNAs, e.g. Rgs4 and Calm3, in rat primary hippocampal neurons in detail. First, we show that Stau2 deficit decreases the dendritic localization of the two target mRNAs. Second, we demonstrate that Stau2 function is mediated by a retained intron in the Calm3 3-UTR. Third, we report that neuronal activity substantially increases dendritic localization of both mRNA targets. In a parallel live cell imaging approach, we investigated the dendritic transport of Rgs4 mRNA in hippocampal neurons. We show that the Rgs4 3-UTR containing MS2 reporter RNA localizes as the endogenous mRNA. Importantly, we discovered a 3-UTR dependent transport bias towards distal dendrites (anterograde direction). Finally, we show transport of the MS2 Rgs4 reporter mRNA to the vicinity of synapses, using dual color imaging with the synaptic marker PSD-95-tagRFP. Taken together, our data provide novel insight at the mechanistic level to our understanding of Stau2-dependent dendritic mRNA transport, localization and expression. This represents significant progress in our long-term goal to unravel how individual synapses might undergo molecular, morphological and functional reorganization upon learning and memory.