Publication Date

2015-06-29

Availability

Open access

Embargo Period

2015-06-29

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Neuroscience (Medicine)

Date of Defense

2015-06-16

First Committee Member

Claes Wahlestedt

Second Committee Member

Nagi Ayad

Third Committee Member

Vance Lemmon

Fourth Committee Member

Richard Jope

Fifth Committee Member

Courtney Miller

Abstract

CGG repeat expansions in the Fragile X mental retardation 1 (FMR1) gene are responsible for a family of associated disorders characterized by either intellectual disability and autism (Fragile X Syndrome, FXS), or adult-onset neurodegeneration (Fragile X-associated Tremor/Ataxia Syndrome, FXTAS). However, the FMR1 locus is complex and encodes several long noncoding RNAs (lncRNAs), whose expression is altered by repeat expansion mutations. The role of these lncRNAs is thus far unknown; therefore we investigated the functionality of FMR4, which we previously identified. “Full”-length expansions of the FMR1 triplet repeat cause silencing of both FMR1 and FMR4, thus we are interested in potential loss-of-function that may add to phenotypic manifestation of FXS. As cis- regulation of FMR1 by FMR4 was not detected, we sought trans-regulated FMR4 targets in vitro. Gene expression and chromatin immunoprecipitation microarrays identified differentially expressed FMR4-responsive genes. Activities of the polyadenylated and chromatin-associated FMR4 in human neural precursor cells (hNPCs) included regulation of genes involved in cell cycling, differentiation, the ubiquitin-proteasome pathway and a G-protein coupled receptor subunit. FMR4 is expected to share a bidirectional promoter with FMR1, but expression of the two transcripts diverges during peak hNPC proliferation. FMR4 decreases, while FMR1 RNA increases, and these changes are accompanied by corresponding differential expression of FMR4 target genes. By regulating gene expression, FMR4 may promote cellular proliferation rather than differentiation, a role supported by S-phase marker assays. We also identified protein partners of FMR4 using MS2-tagged RNA-immunoprecipitation and mass spectrometry. We therefore propose that FMR4’s function is as a gene-regulatory lncRNA and that this transcript may function in normal development. Closer examination of FMR4 increases our understanding of the role of regulatory lncRNA and the consequences of FMR1 repeat expansions.

Keywords

Fragile X Syndrome; FXTAS; long noncoding RNA; lncRNA; neurodevelopment; epigenetics

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