Dissertations, Theses, and Capstone Projects

Date of Degree

9-2024

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Frida Kleiman

Committee Members

Anjana Saxena

Maria Figueiredo Pereira

Moira Sauane

Columba De La Parra

Xavier Grana-Amat

Subject Categories

Biology

Abstract

BRCA1, a gene encoding for the tumor suppressor protein BRCA1, is involved in DNA repair as well as many other important cellular processes. BRCA1, together with BARD1 (BRCA1 Associated RING Domain 1) also functions as an E3 ubiquitin (Ub) ligase. BRCA1 mutation in the RING domain, which is responsible for E3 ligase activity, is associated with increased risk of developing breast cancer. Despite extensive research on the activities of BRCA1, the E3 ligase activity and implications of this activity in cancer are not well understood. This dissertation examines the ubiquitination of the RNA binding protein Human antigen R (HuR) by BRCA1/BARD1 and the implications of this ubiquitination event on gene expression in breast cancer.

HuR binds to mRNA targets in the nucleus and escorts them to the cytoplasm, typically stabilizing them and, hence, regulating gene expression. Oligomerization of HuR is necessary for proper interaction with and stabilization of these mRNA targets. HuR targets are involved in important processes such as cell proliferation, apoptosis, carcinogenesis, and DNA damage response (DDR). Cytoplasmic HuR is correlated with more severe forms of breast cancer, and HuR is a potential target for breast cancer therapies. Previous studies have shown that non- degradative ubiquitination of HuR causes detachment from target transcripts. Additionally, data from our lab showed that BRCA1/BARD1 can ubiquitinate HuR. However, the effects of this ubiquitination on HuR sub-cellular localization, HuR oligomerization, and transcript binding and stabilization have not been established yet.

In this dissertation, I show that HuR is ubiquitinated by BRCA1/BARD1 at Lysine 313 (K313) in the RNA recognition motif 3 (RRM3), and that HuR localization in chromatin increases when ubiquitination at K313 is compromised. Furthermore, my studies indicate that the UV- induced increase in cytoplasmic HuR observed in breast cancer cells with functional BRCA1 is lost in cells with compromised BRCA1-mediated ubiquitination. Furthermore, BRCA1/BARD1- mediated ubiquitination of HuR at K313 alters HuR oligomerization and its binding to target transcript CDKN1A, and preliminary data indicates CDKN1A stability is also affected. Together, these findings elucidate a new pathway through which BRCA1 mutations may lead to transcriptome dysregulation and alter gene expression in breast cancer patients, influencing disease outcomes and therapy success.

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