Localization and local maturation of precursor microRNAs in axons of sensory neurons
Date
2018
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Publisher
University of Delaware
Abstract
Subcellular localization of messenger RNAs (mRNAs) is a conserved mechanism that enables highly polarized cells such as adult mammalian neurons to exert spatial and temporal control over protein synthesis. In neurons, hundreds, if not thousands, of mRNAs are transported into distal axons and undergo local translation. Intra-axonal protein synthesis has been implicated in axon growth and pathway finding during development and rapid injury response and regeneration in adult neurons. Although it is now clear that localized axonal protein synthesis plays an important role in regenerating axons, there has been little understanding of how these localized mRNA translation is temporally regulated. ☐ MicroRNA (miRNA) is a major class of small non-coding RNAs that acts as a regulator of gene expression at the post-transcriptional level. Over the past decade, miRNA-mediated regulation of gene expression has been gaining functional importance in neurons. Recent studies have shown that specific miRNAs are highly enriched in distal axons. These axonally enriched miRNAs could serve as molecular switches for temporal regulation of local protein synthesis. To understand how axon-specific miRNAs respond to nerve injury, I conducted deep sequencing studies using purified axoplasmic RNA from the proximal nerve stump of injured rat sciatic nerve. My sequencing studies identified 141 different miRNAs in rat sciatic nerve axoplasm. Sixty-three of these miRNAs showed significant differential changes in their levels at five time points following nerve injury compared to uninjured sham operated controls. Bioinformatics analysis of predicted axonal mRNA targets of the differentially altered miRNAs revealed that their potential targets were involved in numerous neurological functions in ER response, cytoskeleton dynamics, vesicle formation, neuro-degeneration and-regeneration. These results suggest a vital role of axoplasmic miRNA in the regenerative response for the first time. ☐ Although the role of miRNA-mediated regulation of intra-axonal protein synthesis is beginning to emerge, the mechanism underlying the translocation of specific miRNAs into distal axons remains unknown. Our lab and others showed that both miRNA precursors (pre-miRNAs) and protein components of their processing machinery such as Dicer and KH-type splicing regulatory protein (KSRP) were localized to axons. Based on these findings, I hypothesized that a subset of pre-miRNAs were selectively localized to distal axons, where they were subsequently processed to give rise to biologically active mature miRNAs that function to regulate specific intra-axonal mRNA translation. My results showed that the terminal loop region of axonally localizing pre-miR-433 structure contained a cis-element(s) that strongly governed the localization of RNA to distal axons. Using a compartmentalized culture system, I further demonstrated that selective pre-miRNAs (including pre-miR-433) were processed into the corresponding mature miRNAs in the axonal compartment independent of the cell body. To determine the biological function of mature miR-433-3p, I performed gain of function studies through transient transfection of miR-433-3 mimic in cultured DRG neurons. My results showed that miR-433-3p negatively regulated the expression of Ran binding protein 1 in the growth cone leading to a significant decrease in axonal length. ☐ Altogether, the studies presented in this dissertation provide new insight into the mechanism of differential expression of miRNAs via active transport of the pre-miRNAs, which consequently are processed to corresponding miRNAs and their neurological roles in axon regeneration following injury.
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Keywords
Biological sciences, Axon, MicroRNA, Neuron, Regeneration