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Theses Doctoral

Function and Regulation of ALS/FTD-associated RNA Binding Protein FUS

Tsai, Yueh-Lin

Fused in Sarcoma (FUS) is a nuclear RNA binding protein functioning in a number of essential cellular processes such as RNA processing and DNA damage response. Mutations in FUS gene contribute to 5% of familial Amyotrophic Lateral Sclerosis (ALS) characterized by FUS protein cytoplasmic aggregation. Despite efforts have been made in the past decade, mechanisms of FUS aggregates to induce cytotoxicity are not fully understood. In addition, wild-type FUS protein has been found mis-localized to cytoplasm in sporadic ALS and Frontotemporal Dementia (FTD) patients with unclear mechanisms. Here, we aimed to address the functional consequences of ALS mutant FUS aggregation and investigate the mechanisms of wild-type FUS cytoplasmic translocation. This dissertation is divided into three parts: In the first part, we review pathophysiological mechanisms of FUS and other ALS mutant genes which induce cell death via disrupting six major cellular processes: mRNA processing, non-sense mediated decay, mitochondrial functions, nucleocytoplasmic transport, autophagy and DNA damage response.

In the second part, we aimed to understand the functional consequences of RNA sequestration by FUS aggregates. We performed RNA immunoprecipitation against exogenous or endogenous FUS in the transfected cell lines and mutant FUS ALS patient fibroblasts to isolate RNAs associated with wild-type or ALS mutant FUS. Next, we analyzed the isolated RNAs using poly(A+) RNA-specific sequencing 3’READS and RT-qPCR, and we found many nuclear-encoded respiratory chain complex mRNAs are top-enriched transcripts associated with ALS mutant or overexpressed wild-type FUS. We further demonstrated that respiratory chain complex mRNAs are sequestered in mutant FUS cytoplasmic aggregates and the encoded protein expression levels are suppressed. Finally, we showed that knockdown of respiratory chain complex proteins encoded by FUS-sequestered transcripts can recapitulate mitochondrial dysfunction observed in FUS-transfected cell lines. Our findings in the second part thus provides a novel mechanism by which ALS mutant FUS, as well as overexpressed wild-type FUS, to induce mitochondrial dysfunction via preferential sequestration of respiratory chain complex mRNAs.

The third part focuses on understanding pathways affecting FUS nucleocytoplasmic distribution. By using pharmacological treatments and immunofluorescence, we found that nuclear RNA transcription, export and decay substantially modulate nucleocytoplasmic distribution of wild-type FUS protein. Moreover, we report that FUS antibodies used in immunofluorescence significantly affect the results of nucleocytoplasmic ratio quantification. Intriguingly, we observed altered serine-2/-5 phosphorylation on RNAPII CTD as well as reduced number of nascent transcripts in sporadic ALS patient cells, indicating aberrant transcriptional activity related to cytoplasmic accumulation of nuclear RNA binding proteins. Our findings in the third part provide insights to the importance of nuclear RNA metabolism in modulating FUS localization. We also addressed the inconsistent results reported in previous studies regarding FUS nucleocytoplasmic distribution in response to stress. Altogether, these findings suggest proof-of-principle mechanisms of FUS toxic function and aberrant localization linked to ALS and FTD disease spectrum.

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More About This Work

Academic Units
Biological Sciences
Thesis Advisors
Manley, James L.
Degree
Ph.D., Columbia University
Published Here
April 19, 2021