Defense Date

6-13-2013

Graduation Date

Summer 2013

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

Committee Chair

Mihaela Mihailescu

Committee Member

Jeffry Madura

Committee Member

Michael Cascio

Committee Member

Subha Das

Keywords

FMRP, Fragile X syndrome, G-quadruplex, mRNA

Abstract

Fragile X syndrome, the most common form of inherited mental impairment in humans, affects 1 of 4000 males and 1 of 8000 females. It is caused by the absence of the fragile X mental retardation protein (FMRP), resulting from a CGG trinucleotide repeat expansion in the 5'-untranslated region (UTR) of the fragile x mental retardation-1 (FMR1) gene, and subsequent translational silencing of FMRP. FMRP, a proposed translational regulator of neuronal messenger RNA (mRNA) targets, has three RNA binding domains: two K-homology domains (KH1 and KH2) and one arginine-glycine-glycine (RGG) box domain. FMRP RGG box has been shown to bind with high affinity to G-quadruplex forming mRNAs. G-quadruplexes are formed by stacked G-quartets bonded by Hoogsteen base pairing and stabilized by monocations. FMRP undergoes alternative splicing, including the alternative splice site at exon 15, giving rise to FMRP minor isoforms, truncated within close proximity of the RGG box domain. The binding of FMRP to a proposed G quadruplex structure in the coding region of its own mRNA named FBS has been proposed to affect mRNA splicing events for FMRP minor isoforms. In this study we used biophysical methods to directly demonstrate the folding of FMR1 FBS into two specific G-quadruplexes and analyze its binding by the FMRP isoforms. Additionally, we analyzed the binding of an FMRP mutant in which Ser500 was replaced with Asp500 (ISOP), mimicking FMRP phosphorylation. We showed that the minor splice isoforms bind more tightly to the FBS mRNA, suggesting a negative feedback loop of FMRP binding to its mRNA to regulate alternate splicing.

FMRP associates directly with the Ago1 protein, a key component in the microRNA (miRNA) pathway. Interestingly, one of the FMRP mRNA targets, the microtubule associated protein 1B (MAP1B) mRNA, has a G-quadruplex structure in its 5'-UTR shown to be bound by the FMRP RGG box, and a potential binding site for the miRNA let-7b in its 3'-UTR. In this study we investigated the binding of the let-7b miRNA to this sequence within MAP1B mRNA by using biophysical methods. Dr. Yue Feng at Emory University confirmed the translation regulation of let7b miRNA on MAP1B mRNA.

Format

PDF

Language

English

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