Local processing of small non-coding RNAs

Similar to axonal mRNAs, some miRNAs are differentially distributed in subcellular regions of neurons and their levels are altered following nerve injury. I hypothesize that a subset of miRNAs present in axons is derived from local maturation of pre-miRNAs that are specifically targeted to their final destinations to actively regulate local mRNA translation. We will address the possibility that the levels of mature and pre-miRNAs are altered locally in rat sciatic nerves following injury using an in vitro compartment cell culture system we optimized to separate distal axons from the cell bodies (Figure below). Using the strenuous approach that we have been utilizing we will determine whether axonal pre-miRNAs are locally processed into corresponding mature miRNAs using quantitative RT-PCR (qRT-PCR) (Figure below). We will also use live cell imaging of DRG neurons transfected with synthetic pre-miRNAs covalently labeled with a fluorophore and a quencher, to continuously visualize the dynamic process of pre-miRNA maturation after single-axon axotomy. When the pre-miRNA is processed to a mature miRNA, the fluorophore and quencher will be separated and a fluorescent signal will be emitted.

Local processing of axonal pre-miRNA in isolated axons. Top. Schematic of an in vitro compartment cell culture system for isolation of axons. Bottom. Relative levels of pre- and mature miRNA-148b in the isolated axons. Data (mean ± SD, n=3) are normalized to the levels at 0 h