Date of Award

2021

Document Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Scott A. Barbee

Second Advisor

Nancy Lorenzon

Third Advisor

Schuyler Van Engelenburg

Fourth Advisor

Joseph Angleson

Keywords

Molecular biology, Cellular biology

Abstract

Fragile X Syndrome (FXS) is a neurodevelopmental disorder caused by the disruption of Fragile X Mental Retardation Protein (FMRP) function in neurons, affecting nearly 1 in 7,500 individuals. Although FXS typically occurs from a complete loss of FMRP expression due to a CGG trinucleotide expansion within the 5’UTR of the FMR1 gene, single nucleotide polymorphisms (SNPs) within the KH domains of FMRP have been shown to severely disrupt FMRP function. FMRP is an RNA-binding translation repressor that interacts with ~4% of the neuronal transcriptome. Many target mRNAs encode for proteins important for regulating synaptic processes and modulate synaptic plasticity. It is likely that FMRP differentially regulates this large subset of mRNAs via its association with specific membraneless organelles (MLOs), or granules, that are each involved in regulating different processes of the transcript lifecycle. How FMRP forms and interacts with different MLOs however, is largely unknown. Here we show that multivalent interactions via the two canonical KH domains, KH1 and KH2, and the C-terminal intrinsically disordered region (IDR) function cooperatively to promote FMRP granule formation in Drosophila S2 cells.

Two mutations within the KH domains of FMRP have been linked to severe forms of FXS. We were interested in determining whether these mutations disrupted the

formation or function of FMRP-containing MLOs. Here we studied these missense point mutations, by making the orthologous mutations in the fly KH1 (Gly269Glu) and KH2 (Ile307Asn) domains. Within FRAP experiments of fly S2 cells we found that each of the KH point mutants destabilized the dynamic mobile fraction of FMRP granules, while having no impact on immobile fractions. The KH1 mutant in particular has an important function in granule formation and FMRP association with other MLOs involved in posttranscriptional regulation including stress granules and RNA Processing-bodies. Additionally, we found that the KH1 mutation is defective in FMRP-mediated translation, while the KH2 mutant has no effect.

We also studied the impact of these mutations in Drosophila primary motor neurons (MNs) where FMRP associates with neuronal RNA transport granules (NGs). Within NGs FMRP is thought to translationally repress transcripts during their active transport from the soma out to the synapse. Interestingly, we found that the KH1 mutant severely disrupted the nucleation of FMRP-positive NGs. The KH2 mutant on the other hand destabilized NGs, impacting NG transport out in neurites. Interestingly, we found that these mutations had no impact on camkii transport, a well characterized FMRP target, suggesting that FMRP-NG association and RNA transport may not be functionally linked processes.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Provenance

Received from ProQuest

Rights holder

Emily L. Starke

File size

155 pgs

File format

application/pdf

Language

en

Discipline

Molecular biology, Cellular biology

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