ORCID

http://orcid.org/0000-0002-5593-9125

Date of Award

Spring 5-15-2023

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The regulation of gene expression is essential for all domains of life. Control of gene expression occurs during each of the three steps of mRNA translation into protein. The fidelity and rate of the third step of translation, elongation, has been established as a key regulator of gene expression through its effects on mRNA stability and, thus, protein abundance. Consequently, studies focusing on factors that affect mRNA stability, including mRNA sequence composition and structure, have become integral to understanding the role of translational control in gene regulation. In Chapter 1, I discuss mechanisms of translational control, focusing on the gene regulatory mechanism of polyA tracks. PolyA tracks, defined as at least 11 consecutive adenosines in a 12-nucleotide sequence (12A-1 pattern ), are conserved, intrinsic translational regulatory structures existing in the open reading frame of 2% of human genes. These structures act as "slippery sequences," causing ribosomal frameshifting and stalling during elongation. Both processes trigger mRNA surveillance mechanisms, destabilizing the mRNA and causing a decrease in protein amount. The attenuation of translation caused by polyA tracks is proportional to the number of adenosine nucleotides residing within the regulatory sequence. Previous studies have shown that hypomorphic mutants with predictable levels of protein output can be created by inserting polyA tracks of varying lengths in the ORF (Open Reading Frame) of reporter genes. In Chapter 2, I address how this technology can be expanded in reporter genes encoding both secretory and membrane proteins. Our results show that the insertion of polyA tracks in the open reading frame of the secretory protein interleukin-2 (IL2) and membrane protein CD20 leads to a predictable reduction in protein expression and functionality that is unaffected by the presence of a signaling sequence. Secretory and membrane proteins compose a large percentage of the proteome and are involved in diverse fundamental biological pathways. Thus, creating hypomorphic mutations in these gene types expands the knowledge of their biological significance. Furthermore, in Chapter 2, I will discuss the use of CRISPR Cas9 for the endogenous insertion of polyA tracks of various lengths in the opening reading frame of the tumor protein P53 (TP53) and the AU-rich element RNA-binding protein 1 (AUF1). Like creating hypomorphic mutants using polyA track technology in reporter genes, CRISPR/Cas9 genetic editing in conjunction with polyA track insertion creates stable, predictable hypomorphic mutants. Through the creation of endogenous CRISPR/Cas9-engineered TP53 and AUF1 hypomorphic mutants, the effects of various levels of decreased gene expression of both genes on their respective downstream targets were analyzed. Insertion of polyA tracks into reporter genes or endogenous gene insertion using CRISPR/Cas9 provides an effective and invaluable tool for creating hypomorphic mutants. Moreover, the primary focus of this study is to elucidate the biological significance of polyA track-containing genes. In Chapter 3, I will narrow my focus to characterizing the biological mechanism and clinical relevancy of the polyA track-containing gene ZCRB1 (zinc finger CCHC-type and RNA-binding motif 1)--a vital component of the minor spliceosome. The functional gene regulatory role of polyA tracks can be demonstrated by examining stabilizing A>G and destabilizing G>A synonymous mutations in the polyA tracks of ZCRB1. Several destabilizing and frameshift mutations within the polyA track of ZCRB1 have been found in cancer. Preceding this study, it was unknown what effect these mutations may have on the functionality of ZCRB1. Our proteomic analysis implies that the destabilizing and frameshift-inducing ZCRB1 K137K (411G>A) mutation may affect the protein interactions of ZCRB1. As abnormal forms and levels of protein may negatively impact cellular health, leading to cancer, further functional characterization of polyA track mutations in ZCRB1 will broaden the understanding of how gene regulation and protein production are altered in cancer. In addition to characterizing the role of ZCRB1 in the minor spliceosome, I present the role of ZCRB1 in regulating NMD (Nonsense-Mediated Decay) and other polyA track-containing genes. This finding argues for complex feedback over a regulation loop. Furthermore, as described in Chapter 3, a significant result of this study shows that 14% of polyA track genes are primarily regulated by NMD. These findings add to the mechanistic understanding of how polyA tracks regulate gene expression. Previous studies have implied a more considerable contribution of ribosomal stalling and induction of the NoGo Decay mRNA surveillance mechanism. Together these findings support the necessity of a continued understanding of the role of polyA tracks in regulating gene expression and the functional effect they present to the genes that contain them.

Language

English (en)

Chair and Committee

Sergej Djuranovic

Committee Members

Joseph D. Dougherty, Jacqueline E. Payton, Hani S. Zaher, Timothy Schedl,

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