Fragile X related proteins: mapping distinct domains controlling axonal localization, RNA granule assembly and fibril formation

Stackpole, Emily

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Year:: 2014
Contributor:: Stackpole, Emily (creator); Fallon, Justin (Director); Hart, Anne (Reader); Barnea, Gilad (Reader); Moore, Melissa (Reader); Brown University. Neuroscience (sponsor)
Genre:: theses
Subject:: translation; FXR2P; fibril; myristoylation; Axons; RNA
Extent:: xiii, 149 p.
DOI: https://doi.org/10.7301/Z0NS0S7J

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Abstract:: The exquisite morphology of neurons requires responses to stimuli in a spatially regulated manner. One means to achieve spatially-restricted control is through local translation, where mRNAs are transported to specific sites and translated in response to local cues. In axons of the mammalian brain, the RNA binding protein FMRP and its homologs FXR1P and FXR2P localize to specialized RNA granules, termed Fragile X granules (FXGs), which are poised to regulate translation in a synapse specific manner. FXR2P is a component of all FXGs and is necessary for their presence. This thesis therefore characterized the mechanisms that regulate FXR2P localization within axons and promote FXR2P incorporation into RNA granules. I first demonstrate that FXR2P is N-myristoylated, classifying it as a lipid-modified RNA binding protein. Using transfection of cortical neuron cultures, I show that N-myristoylation of FXR2P is not required for localization to axonal granules, but instead acts to regulate its distribution within axons. I next determined which domains of FXR2P contribute to RNA granule assembly by performing an unbiased insertional mutagenesis screen and characterizing 18 full-length EGFP-FXR2P fusions. Using primary neurons, I found that FXR2P with insertion of EGFP into specific sites in the low complexity (LC) domain formed distinct types of highly ordered fibrils. Inserts at aa416 formed curvilinear structures termed Type I FXR2P fibrils. Ultrastructural analyses showed Type I fibrils are present individually and in bundles, both of which associate with ribosomes. FXR2P with EGFP inserted at either aa435 or aa439 formed apparently rigid Type II FXRP fibrils that are also present individually or in fibril bundles. By electron microscopy, I found that while ribosomes are associated with individual Type II fibrils, they do not associate with fibril bundles. Furthermore, I demonstrate that the RNA-binding KH2 domain of FXR2P cooperates with its LC domain to promote fibrillization in neurons. Finally, I demonstrate that the LC domain of FXR2P, but not the KH2 domain, is required for the RNA granule assembly. Together this work begins to elucidate the mechanisms that FXR2P employs to form RNA granules and target these complexes within axonal domains.
Notes:: Thesis (Ph.D. -- Brown University (2014)

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Rights: In Copyright
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Citation

Stackpole, Emily, "Fragile X related proteins: mapping distinct domains controlling axonal localization, RNA granule assembly and fibril formation" (2014). Neuroscience Theses and Dissertations. Brown Digital Repository. Brown University Library. https://doi.org/10.7301/Z0NS0S7J

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Neuroscience Theses and Dissertations

Theses and Dissertations for the Neuroscience department.
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