Molecular mechanisms underlying post-transcriptional regulation of axonally localized mRNAs.
Interaction of cis-localization elements and trans-acting factors modulates the axonal/dendritic transcriptome including non-coding RNAs:
Hundreds of transcripts are known to localize into axons and our recent studies clearly demonstrate that specific non-coding RNAs are enriched in distal processes of neurons. However, we have little mechanistic understanding of how the transport and local translation is regulated at the level of individual mRNAs. I hypothesize that RNA-binding protein (RBP)/RNA interactions will determine dynamics of the local transcriptome and the newly synthesized proteome for efficient neuronal function. To test this hypothesis, we will identify the RBPs that bind to the cis-elements within UTRs of the localizing RNAs using RNA electrophoretic mobility shift assays (REMSA) (Figure below) and proteomics. We previously showed that mRNAs compete for binding to shared RBP for axonal localization and stability. I hypothesize that different levels of local transcriptome and proteome are determined by the affinity of RNAs for and expression levels of their RBPs. We have significant expertise to study the binding mechanisms of RNA-RBP association.
Representative agarose gel from REMSA analyses resolving RNA/protein complexes. (Right) 10 nM Cy5.5-tagged Gap43 or IR800-tagged Nrn1 AREs (AU-rich regions) were bound to 200 nM HuD-GST and then competing ARE was added. RNA/protein complexes (arrows), unbound AREs (arrowheads). (Left) Fluorescence polarization absorption (FPA) assay for Gap43 (red) and Nrn1 AREs (green) binding to increasing concentrations of HuD-GST was used to determine the value of Kd.