Date of Degree

9-2021

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Frida E. Kleiman

Committee Members

Patricia Rockwell

Alejandra Alonso

Jimmie E. Fata

Diego Loayza

Patricia Silveyra

Subject Categories

Biochemistry | Molecular Biology

Keywords

mRNA 3' end processing, nuclear deadenylation, estrogen signaling, estrogen receptor alpha, gene expression

Abstract

Estrogen signaling regulates various physiological processes within the female reproductive, cardiovascular, immune, and central nervous systems and is predominantly mediated by estrogen receptors (ER), ERα and ERβ. Transcriptional regulation by ERs has been widely studied, where activated ERs bind either directly to target genes through binding to estrogen response elements or indirectly through interacting with other proteins, resulting in changes in gene expression. However, the potential role of factors involved in the estrogen signaling pathway in posttranscriptional regulation has not been extensively documented. As 70-80% of breast cancers (BCs) are identified as ER(+), studying how these factors, particularly ERα, regulates the transcriptome and gene expression through its role in messenger RNA (mRNA) 3' end processing will help us further understand the unique profiles of different BCs. The results in this dissertation reveal a novel role of ERα in mRNA 3’ processing. mRNA 3’ end processing plays an important role in balancing the biosynthesis and degradation of mRNAs under different cellular conditions, thereby affecting steady-state levels of cellular mRNAs and contributing to the control of gene expression. Removal of the poly(A) tail at the 3’ end of mRNAs, a process referred to as deadenylation, is regulated by the interaction between tumor suppressor p53 and poly(A)-specific ribonuclease (PARN), the major deadenylase identified in the nucleus of mammalian cells, under normal and DNA damage response (DDR) conditions. Interestingly, as studies have shown feedback loops of p53 with either PARN and/or ERα, I propose that the interplay between these factors may affect the regulation of mRNA 3’ end formation of target genes. By either depleting ERα expression using small-interfering RNA (siRNA) or inhibiting its function using fulvestrant, a selective estrogen receptor downregulator, my results indicate that ERα is an activator of nuclear deadenylation in luminal A (ERα+) MCF7 BC cells. Furthermore, I show that this effect on deadenylation is dependent on PARN deadenylase by performing in vitro deadenylation assays using a capped/radiolabeled L3(A30) RNA substrate with recombinant proteins. Interestingly, co-immunoprecipitation assays reveal (a) complex(es) formation between ERα, PARN, and p53. Supporting these findings, analysis of samples from BC cells with different expression patterns of ERα and p53, such as in luminal A (ERα+) T47D or triple negative BC (TNBC) MDA-MB-231 cells, confirm that ERα plays a role in activating PARN-mediated deadenylation under non-stress conditions. Moreover, analysis of published genome-wide studies that identified mRNA targets of ERα or PARN allowed for the recognition and validation of KLHL24, ID1 and LUM as common mRNA targets for both factors in MCF7 cells, further supporting the functional overlapping of ERα and PARN in regulating transcript levels in a transactivation-independent manner. KLHL24 encodes for a cullin 3–RBX1 ubiquitin ligase substrate receptor, which is expressed in breast mammary tissue but its function in this tissue has not been extensively studied. LUM encodes for lumican, a proteoglygan whose expression is abundant in the extracellular matrix of various tissues, including breast, and is thought to be a key regulator for epithelial to mesenchymal transition. ID1 encodes for an inhibitor of DNA binding 1 protein and it has been shown to play important roles in cell differentiation, tumor angiogenesis, cell invasion, and metastasis, particularly in promoting lung metastasis in BC. Although further studies are needed to identify additional mRNA targets, these results suggest that steady-state levels of specific common mRNAs might be affected by ERα expression through its role in nuclear deadenylation. Together, my studies provide new insights into posttranscriptional regulation of gene expression by ERα. As most BCs are identified as ER(+), studying how factors within the estrogen signaling pathway, specifically ERα, regulates the transcriptome through its role in mRNA 3' end processing will help us understand unique profiles of BCs and contribute to identifying new molecular targets and/or biomarkers for treatment development for different BCs.

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