Year of Publication

2015

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Molecular and Cellular Biochemistry

First Advisor

Dr. Haining Zhu

Abstract

FUS is an RNA binding protein implicated in the motor neuron disease— amyotrophic lateral sclerosis (ALS, also called Lou Gehrig’s disease). ALS is a fatal neurodegenerative disease characterized by progressive motor neuron death. Mutations in the FUS gene cause about 4% of familial ALS (FUS ALS). Mutated FUS protein mislocalizes from the motor neuron nucleus to the cytoplasm and forms inclusions in the cytoplasm. It is unclear how FUS mislocalization induces motor neuron dysfunction and degeneration. This dissertation research was designed to investigate the physiological functions of FUS in the nucleus, with a purpose to shed light on the pathogenesis of FUS ALS. Using biochemical and cell biology approaches, we revealed that there are two functionally distinct pools of FUS inside the nucleus. A portion of FUS is bound to active chromatin domains and is involved in gene transcription regulation. ALS mutations significantly decrease FUS chromatin binding. We further discovered chromatin binding requires FUS oligomerization, which is mediated by an intrinsically disordered QGSY (glutamine-glycine-serine-tyrosine) -rich region in FUS.

Using confocal microscopy and an in vitro FUS oligomerization assay, we identified chromatin-associated nuclear RNAs as the trigger of FUS oligomerization. We further discovered that the RNA binding ability of FUS is also required for the cytoplasmic inclusion formation, which does not require the QGSY-rich region. By exchanging localizations of wild-type FUS and mutant FUS, we demonstrated that subcellular localization and RNAs play a more important role than ALS mutations in determining distinct FUS distribution and organization in different cellular compartments.

By knocking down protein arginine methyltransferase gene and using methylation inhibitor treatment, we found that a post-translational modification of FUS—arginine methylation—can regulate FUS chromatin binding. Suppression of arginine methylation restored mutant FUS binding to active chromatin domains. Altogether, we revealed the distribution-related FUS physiological functions in the nucleus and identified a potential way to reverse the destructive effect of ALS mutations on wild-type FUS.

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