Date of Award
Summer 2023
Document Type
Open Access Dissertation
Department
Biological Sciences
First Advisor
Jeffery Twiss
Abstract
Neurons are highly polarized cells with expansive cytoplasmic processes, axons and dendrites, which create unique subcellular domains. Localization and translation of mRNAs in these subcellular regions is now recognized as a critical mechanism through which polarized cells establish and maintain the specialized domains that are needed for their functions. RNA profiling studies have uncovered hundreds to thousands of mRNAs in axons and these axonal mRNA profiles differ across neuronal populations, physiological conditions, and developmental growth states. Functionally organizing these mRNA populations into cohorts of coregulated mRNAs via RNA binding proteins allows us to better understand how the transcriptome is regulated as a whole. In this dissertation, I focus on growth associated mRNAs, Cdc42, RhoA and Calr, and the regulation of their axonal localization and subsequent translation.
CDC42 is a small Rho GTPase that, when activated, signals through downstream effectors triggering actin polymerization, which in neurons can be associated with axon growth. The Cdc42 gene undergoes alternative splicing that generates two mRNA variants with different 3’ untranslated regions (UTRs) that encode two distinct proteins with different C-termini: palm-CDC42 with exon 6 as its terminal exon and prenyl-CDC42 that excludes exon 6 ending in exon 7. My results show that prenyl-Cdc42 mRNA preferentially localizes into axons while palm-Cdc42 mRNA is restricted to the somatodendritic region of CNS and PNS neurons. Axonally localized prenyl-Cdc42 mRNA increases axon growth, but this also requires prenylation of the encoded protein. Prenyl-CDC42 and another Rho family member, RHOA, show reciprocal regulation of their axonal mRNA levels and translation in response to the growth-inhibiting stimulus aggrecan. This reflects the antagonistic effects of CDC42 and RHOA activities on actin microfilaments. Interestingly, nt 801-875 of prenyl-Cdc42 mRNA is AU-rich, and I find that it is a target for the RNA binding protein KHSRP, which is known to promote mRNA decay through the cytosolic exosome. Prenyl-Cdc42 mRNA is acutely degraded in axons in response to aggrecan through a mechanism which requires axoplasmic Ca2+ and KHSRP. KHSRP deficient mice, which show accelerated axon regeneration, have approximately five-fold more axonal prenyl-Cdc42 mRNA than wild type mice.
Initial increases in axoplasmic Ca2+ following axotomy must be buffered for regeneration to proceed. Calr mRNA encodes an ER-resident Ca2+-buffering chaperone protein. It has been shown that Calr mRNA localizes into axons and that Ca2+ elevation activates its translation locally, but we have not known what factors drive its transport into and translation within axons. Calr mRNA contains two 3’UTR RNA localization motifs and one 5’UTR translational regulation motif. To identify proteins that bind to these motifs, I used RNA affinity mass spectrometry. This identified that many known RNA binding proteins can bind to the Calr mRNA motifs including PURA, KHSRP, NCL, and HUR. There was some overlap between binding proteins for the 5’ and 3’UTR motifs, and previous studies pointed to an interaction between these regions for translational regulation. My studies show that PURA protein coprecipitates with the distal 3’UTR element of Calr. PURA also colocalizes with axonal Calr mRNA in cultured neurons and depletion of PURA decreases axonal levels of Calr mRNA. While much more research is needed to understand the transcriptome and translatome of axons, these data help to untangle the unknown web that is regulation of axonal localization and translation.
This dissertation includes a supplementary file containing mass spectrometry results for chapter 4.
Rights
© 2023, Matthew Davis Zdradzinski
Recommended Citation
Zdradzinski, M. D.(2023). Molecular Mediators of Axonal Transport and Local Translation Of CDC42, RHOA Molecular Mediators of Axonal Transport and Local Translation of and Calreticulin mRNAs and Their Implications for Axon Growth and Regeneration. (Doctoral dissertation). Retrieved from https://scholarcommons.sc.edu/etd/7404