All ETDs from UAB

Advisory Committee Chair

David A Schneider

Advisory Committee Members

Josh Stern

Karina Yoon

Document Type

Thesis

Date of Award

2021

Degree Name by School

Master of Science (MS) Heersink School of Medicine

Abstract

RNA polymerases are primarily multi-subunit enzymes that synthesize RNAs from template DNA strands. RNA polymerase I (Pol I) is the eukaryotic RNA polymerase that synthesizes the majority of ribosomal RNA (rRNA) for ribosome production. These include the 5.8S, 28S, and 18S rRNAs which are synthesized from a polycistronic gene in the nucleolus. The rRNAs synthesized by Pol I, the 5S rRNA, & ribosomal proteins come together to synthesize ribosomes through ribosome biogenesis. Dysregulation of Pol I activity has been established to contribute to dysregulation of ribosome biogenesis and disease state development. These conditions include but are not limited to Cincinnati Type Acrofacial Dystosis, Burn-McKeown syndrome, and Warsaw Breakage syndrome. Ribosome biogenesis is the highly conserved process responsible for the synthesis of ribosomes. Ribosomes are important enzymes that synthesize proteins from template messenger RNA (mRNA) through translation. Dysregulation of ribosome biogenesis has been established to contribute to disease state development. Increasing the rate of ribosome biogenesis has been established to contribute to conditions such as cancer. Cancer cell proliferation requires an increase in protein production to support the increase in cellular division contributing to rapid cell growth. An increase in ribosome biogenesis increases ribosomes synthesizing proteins which is required for rapid cell growth. iv Multiple mutations were identified within the POLRA gene that encodes the RPA194 subunit of human Pol I from The Cancer Genome Atlas by our collaborators in the Laiho Lab at Johns Hopkins University. Two mutations of interest are the Q1217K and P1222L mutations and they are located within the trigger loop region of Pol I. The trigger loop is a highly conserved region within RNA polymerases and contributes to their overall catalytic activity. These two mutations are of interest because their identification in cancer cells, Pol I’s contribution to ribosome biogenesis, and phenotypes from trigger loop mutations within eukaryotic RNA polymerases. I hypothesize these trigger loop mutations result in a hyperactive Pol I. This would be expressed as an increase in catalytic activity of Pol I resulting in an increased growth rate of cells and rate RNA synthesis. The mutations were characterized biochemically and physiologically within RPA194’s yeast homolog A190 from Saccharomyces cerevisiae. Physiological characterization of the mutations displayed that when they are expressed within Pol I, they are lethal. Biochemical characterization of the P1222L mutation displayed that the mutation renders Pol I inactive. These results support each other and characterization of other trigger loop mutations. An inactive Pol I would result in a failure to synthesize rRNA and this would prevent ribosome biogenesis and result in cell death. These results refute my hypothesis that the mutations would result in a hyperactive Pol I.

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