Dissertations, Theses, and Capstone Projects

Date of Degree

5-2019

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Frida Kleiman

Committee Members

Bin Tian

Diego Loayza

Kevin Ryan

Olorunseun Ogunwobi

Subject Categories

Biology | Cancer Biology | Cell Biology | Genetics | Molecular Biology | Molecular Genetics

Keywords

Alternative Polyadenylation, DNA Damage Response, CDKN1A, Cell Cycle, RNA Processing

Abstract

Cellular homeostasis is achieved by the dynamic flux in gene expression. Post-transcriptional regulation of coding and non-coding RNA offers a fast method of adapting to a changing cellular environment, including deadenylation, microRNA (miRNA) pathway, and alternative polyadenylation (APA). In this dissertation, I explored some of the mechanisms involved in the post-transcriptional regulation of gene expression. The main hypothesis in these studies is that a single APA event after DNA damage is governed by specific conditions and factors outside of current known regulators of APA, and that the resultant transcript has a role in the DNA damage response (DDR). My aims were a) to investigate the RNA processing and coding potential of a CDKN1A APA transcript, b) to elucidate the conditions and factors involved during CDKN1A APA induction, and c) to determine whether the CDKN1A APA transcript possess any biological function.

In Chapter II, I investigated the miRNA-dependent recruitment of PARN nuclear deadenylase to p53 mRNA 3’ untranslated region (3’UTR), as part of the regulatory feedback loop involving PARN and p53. Then my studies focused on the function and regulation of APA during the progression of DDR. Understanding the scope and impact of APA in different cellular settings will help us to understand changes in the transcriptome and proteome and will offer alternative avenues for specific and effective pharmaceutical design. In Chapter III, I present evidence of the magnitude of APA events occurring within introns during DDR, and the mechanisms involved in the regulation of these intronic APA events. In Chapter IV, I further investigate the coding potential and post-transcriptional processing of an intron-APA transcript from the cyclin-dependent kinase inhibitor 1A (CDKN1A) gene, which encodes the cell cycle arrest protein p21. Specifically, I observe that the CDKN1A intron-APA transcript has the potential to be spliced from an alternative splice site, generating a ‘cryptic’ exon upstream of intron-APA site, but also to exist as a non-spliced isoform. My results also indicate that neither isoform is protein-coding, implicating the resultant transcripts are stable long non-coding RNAs. In Chapter V, I characterized the CDKN1A intron-APA transcripts and analyzed the mechanisms involved in their regulation, including the effect of both DNA binding proteins (p53) and RNA binding proteins (HuR). Finally, in Chapter VI, I present evidence that far from being a by-product of transcriptional control of canonical CDKN1A full-length mRNA, intron-APA transcripts from CDKN1A gene play roles both in a non-stress and in DDR conditions, and also can alter the timing of events during DDR. Thus, the data presented in this dissertation provides new insights into the regulation of APA as well as the cellular roles that these events can participate. This offers a greater understanding of DDR, which aids in clearly defining pathways during therapeutic development for diseases, such as cancer.

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