Date of Award

Spring 1-1-2017

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Thomas R. Cech

Second Advisor

Roy Parker

Third Advisor

Rob T. Batey

Fourth Advisor

Karolin Luger

Fifth Advisor

Rui Yi

Abstract

RNA and protein interact with each other in the cell nucleus to form RNP (ribonucleoprotein) complexes. These RNPs play key roles in various steps of gene expression. This thesis focuses on two nuclear RNA-binding proteins: Fused in Sarcoma (FUS) and Polycomb Repressive Complex 2 (PRC2).

The normal functions of FUS are not fully understood, but RNA binding has been suggested to be crucial for FUS function and many RNA targets have been identified. Yet the features of RNAs necessary for FUS binding had not been systematically characterized. FUS bound all five published RNA motifs and other RNAs tightly, including fragments of an Escherichia coli mRNA, suggesting that FUS has a wide range of nucleic-acid binding ability. Additional pull-down experiments reveal that FUS binds the C-terminal domain (CTD) of RNA polymerase II in an RNA-dependent manner, providing insight into its function.

PRC2 has been shown to interact with RNAs promiscuously in vitro and in vivo, as evidenced by the broad spectrum of transcripts that it binds. Detailed quantitative binding experiments with RNAs of defined sequence and length, coupled with analysis of fRIP-seq and ChIP-seq data, show that PRC2 preferentially binds G-tracts in RNA. The G-tracts can be either single-stranded or, preferably, folded G-quadruplexes, which are ubiquitous in the transcriptome. This explains the promiscuous nature of PRC2 targeting to RNA. Additional binding and histone methyltransferase assays showed that RNA is not a methyltransferase inhibitor; rather it sequesters PRC2 away from nucleosomes. More surprisingly, human PRC2 has robust DNA-binding activity, and RNA inhibits PRC2 binding to nucleosome by competing PRC2 off the linker region of nucleosomes.

Collectively, findings presented in this thesis offer new insights into the mechanistic details underlying the recognition of RNAs by FUS protein and the PRC2 methyltransferase. It is clear that the dysregulation of FUS is linked to neurodegenerative diseases, and that PRC2 is dysregulated in some types of cancers. Thus, understanding the mechanism by which these nuclear RNA-binding proteins recognize RNAs helps pave new avenues towards developing novel therapeutics to treat RNA-dominant diseases.

Available for download on Tuesday, August 28, 2018

Included in

Biochemistry Commons

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