Date of Degree

2-2020

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Frida E. Kleiman

Committee Members

Patricia Rockwell

Susan Rotenberg

Hualin Zhong

Anjana D. Saxena

Subject Categories

Biochemistry | Biology | Biotechnology | Molecular Biology

Keywords

RNA processing, RNA binding proteins, post translational modifications, RNA stability

Abstract

Despite our understanding of the biochemistry of the BRCA1/BARD1 E3 ligase, we know very little of the mechanism by which its cellular substrates, how they are chosen, how its E3 Ub ligase activity is regulated, which are the cofactors involved in this regulation, and how the substrates are modified during DDR. In this dissertation, I further evaluate BRCA1/BARD1-mediated E3 ligase activity and how CstF-50 and p97 regulate this reaction working as cofactors. My data show that BRCA1/BARD1 is an E3 ligase that ubiquitinates the RNA binding protein Human antigen R(HuR).

HuR is abundant in the nucleus and is transported to the cytoplasm associated to target mRNAsand this change in subcellular localization appears to be linked to HuR functions. Clinical studies reveal that cytoplasmic HuR is associated with more aggressive forms of breast cancer and is a putative prognostic factor for survival, making HuR a promising drug target for cancer therapy. HuR modulates different aspects of mRNA life, such as transport, translation and stability; through binding to the sequence motifsAU-rich elements (ARE) in the 3’ untranslated region (3’ UTR) of target mRNAs. HuR mRNA targets are involved in carcinogenesis, cell proliferation, and DNA damage response (DDR).Ubiquitination has been implicated in remodeling HuR interaction with target mRNAs as part of the DDR. Non-degradative ubiquitination of HuR signals its dissociation from target mRNAs, such as p21 and MKP-1, resulting in mRNA destabilization under non-stress conditions. While p97, an escort factor of polyubiquitinated substrates with ATPase activity, is involved in this process, the E3 ubiquitin (Ub) ligase responsible for HuR modification has not been identified yet.

The studies presented in this dissertation show that HuR can be ubiquitinated by the tumor suppressor BRCA1/BARD1, and this BRCA1/BARD1-mediated ubiquitination of HuR can be regulated by the mRNA 3’ processing factor CstF-50 and p97.Under non-stress conditions, nuclear HuR binding to ARE sequence in TP53 3’ UTR is diminished by BRCA1/BARD1-mediated ubiquitination resulting in a decrease in TP53 mRNA stability through active deadenylation. Following DNA damage, non-ubiquitnated HuR binds ARE sequence inTP53 3’ UTR increasing mRNA stability and, hence, p53 expression.

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