Date of Award

Spring 2024

Degree Type

Thesis

Department

Biological Sciences

Director of Thesis

Dr. Jeff Twiss

Second Reader

Lauren Vaughn

Abstract

Following traumatic injury, axons in the peripheral nervous system (PNS) can spontaneously regenerate, albeit rather slowly. This regeneration requires messenger RNA (mRNA)-localization into and translation within the axons. One such mRNA originates from the CDC42 gene, which produces two mRNA splice variants: Prenyl-CDC42 and Palm-CDC42 encoding mRNAs. CDC42 promotes axon growth and regeneration by regulating actin filament polymerization in growth cones of axons. This plays an important role to support nerve regeneration in humans. The prenyl-Cdc42 mRNA is found in both central nervous system (CNS) and PNS axons, where it can be locally translated into CDC42 protein, which is subsequently prenylated. In this study, we identified that prenyl-Cdc42 mRNA’s axonal localization is dependent on a short motif within the 3’ untranslated region (UTR), corresponding to nucleotides (nt) 764-800. RNA affinity mass spectrometry (RAMS) using biotinylated synthetic oligonucleotides corresponding to localizing 764-825 and non-localizing 800-875 nt sequences were used as bait to isolate and identify RNA binding proteins for these regions. RNA affinity pulldowns were performed using the localization sequence as bait with PC12 cell lysates to initially validate candidates identified by RAMS. This was later performed using lysates from cultures of dorsal root ganglion (DRG) neurons. The resulting bound fraction was analyzed by western blotting and probing for the specific RNA binding protein targets CCAR1, PTBP3, and MBNL1. CCAR1 and PTBP3 were validated as binding to the prenyl-Cdc42 764-825 nt (and later 764-800 nt). To further test for interaction between the localization motif on prenyl-Cdc42 mRNA with CCAR1 and PTBP3, fluorescence in situ hybridization and immunofluorescence (FISH/IF) was performed using rat DRG neurons. CCAR1 colocalized with prenyl-Cdc42 2 mRNA in axons. siRNA knockdown of CCAR1, but not PTBP3, significantly decreased axonal signal for prenyl-Cdc42 mRNA in DRG axons compared to a non-targeting siRNA. Together, these data indicate that CCAR1 is necessary for axonal localization of prenyl-Cdc42 mRNA and provides new strategies for promoting axonal mRNA localization/translation and accelerating axon regeneration.

First Page

1

Last Page

50

Rights

© 2024, Asley I. Loomis

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