Mechanisms of regulation of mRNA 3\u27 processing by p53 pathway

Although the p53 network has been intensively studied, genetic analyses long hinted at the existence of components that remained elusive. This dissertation focuses on the study of the regulation of mRNA 3\u27 processing during DNA damage response (DDR) by the p53 pathway and the regulation of p53 expression by the mRNA 3\u27 processing machinery. The results in this dissertation revealed new roles of tumor suppressor p53 in mRNA 3\u27 processing. In Chapter II, I showed that p53 inhibits the cleavage step of polyadenylation reaction and that cells with different levels of p53 expression have different mRNA processing profiles. As part of the same response to DNA damage, my results indicate that p53 also activates PARN-dependent deadenylation in the nucleus (Chapter III). In Chapter IV, I demonstrated that p53 mRNA is one of the biological targets of nuclear PARN under non-stress conditions. Extending these studies, in Chapter V, I established that both AU-rich element (ARE) and miR-125b binding site are important for the binding of PARN to the p53 mRNA and activation of p53 pathway. Together these results show a feedback loop between PARN deadenylase and one of its targets, the tumor suppressor p53: While PARN keeps p53 levels low by destabilizing p53 mRNA through ARE- and microRNA-binding sites in non-stress conditions; the increase in p53 levels after UV treatment results in the activation of PARN deadenylase in a transcription-independent manner. As the levels of p53 expression levels increase after DNA damage, the PARN-mediated down-regulation of p53 mRNA should be reverted during the progression of DDR. In Chapter VI, I found that under DNA damaging conditions HuR, a ubiquitously expressed ARE-binding protein, can compete for binding to the p53 3\u27UTR with both PARN and Ago-2, resulting in the release of PARN and Ago-2 from p53 mRNA and the increase of p53 expression levels. Finally in Chapter VII, I analyzed the usage of alternative polyadenylation signals (APA) during DDR. My results indicate that increase in intronic-polyadenylated isoforms of genes involved in DDR occurred after UV treatment, indicating that APA might represent another potential mechanism of controlling gene expression during the response to DNA damage. Together this dissertation provides new insights into p53 function and the mechanisms behind the regulation of mRNA 3\u27 end processing and hence gene expression in different cellular conditions

PARN deadenylase is involved in miRNA-dependent degradation of TP53 mRNA in mammalian cells

mRNA deadenylation is under the control of cis-acting regulatory elements, which include AU-rich elements (AREs) and microRNA (miRNA) targeting sites, within the 3′ untranslated region (3′ UTRs) of eukaryotic mRNAs. Deadenylases promote miRNA-induced mRNA decay through their interaction with miRNA-induced silencing complex (miRISC). However, the role of poly(A) specific ribonuclease (PARN) deadenylase in miRNA-dependent mRNA degradation has not been elucidated. Here, we present evidence that not only ARE- but also miRNA-mediated pathways are involved in PARN-mediated regulation of the steady state levels of TP53 mRNA, which encodes the tumor suppressor p53. Supporting this, Argonaute-2 (Ago-2), the core component of miRISC, can coexist in complexes with PARN resulting in the activation of its deadenylase activity. PARN regulates TP53 mRNA stability through not only an ARE but also an adjacent miR-504/miR-125b-targeting site in the 3′ UTR. More importantly, we found that miR-125b-loaded miRISC contributes to the specific recruitment of PARN to TP53 mRNA, and that can be reverted by the ARE-binding protein HuR. Together, our studies provide new insights into the role of PARN in miRNA-dependent control of mRNA decay and into the mechanisms behind the regulation of p53 expression

Intronic cleavage and polyadenylation regulates gene expression during DNA damage response through U1 snRNA

The DNA damage response involves coordinated control of gene expression and DNA repair. Using deep sequencing, we found widespread changes of alternative cleavage and polyadenylation site usage on ultraviolet-treatment in mammalian cells. Alternative cleavage and polyadenylation regulation in the 3ʹ untranslated region is substantial, leading to both shortening and lengthening of 3ʹ untranslated regions of genes. Interestingly, a strong activation of intronic alternative cleavage and polyadenylation sites is detected, resulting in widespread expression of truncated transcripts. Intronic alternative cleavage and polyadenylation events are biased to the 5ʹ end of genes and affect gene groups with important functions in DNA damage response and cancer. Moreover, intronic alternative cleavage and polyadenylation site activation during DNA damage response correlates with a decrease in U1 snRNA levels, and is reversible by U1 snRNA overexpression. Importantly, U1 snRNA overexpression mitigates ultraviolet-induced apoptosis. Together, these data reveal a significant gene regulatory scheme in DNA damage response where U1 snRNA impacts gene expression via the U1-alternative cleavage and polyadenylation axis

A modified Kiss, Marry, Kill game, a novel game-based learning activity for biology and anatomy and physiology students to learn and retain complex scientific concepts

ABSTRACT This study introduces how a series of fun and interactive discussion board activities can enhance student engagement and understanding of important chemical and biological concepts in college biology and anatomy and physiology courses. The activity is a novel game-based learning approach based on a modified version of the “Kiss, Marry, Kill” (KMK) game, where students choose between course content-related choices provided (e.g., three types of cellular organelles—mitochondria, ribosomes, and nucleus) and explain their choices. By requiring students to evaluate and justify their choices, this method promotes critical thinking and collaborative learning in biology education. Participants also engage in discussions about these choices, fostering collaborative learning. This article details the implementation process, anticipated outcomes, and pedagogical benefits of this innovative approach

Abstract 3188: Nucleolin phosphorylation mediated regulation of gene expression in determining cellular fate during the DNA damage response (DDR).

Abstract The phosphoprotein nucleolin integrates several critical cellular processes, such as cell growth, proliferation, cell cycle arrest, apoptosis as well as DDR. Elevated levels of nucleolin are found in highly proliferative cells including a variety of tumors. Nucleolin is highly phosphorylated at the N-terminus by two major kinases: interphase casein kinase 2 (CK2) and mitotic cyclin-dependent kinase (Cdk). Earlier we have demonstrated that the N-terminus of nucleolin associates with Hdm2 to stabilize p53 protein and causes p53-mediated apoptosis. Additionally, nucleolin via its RNA-binding properties has been demonstrated to regulate the stability of several mRNAs and enhance translation. Besides, nucleolin post-translational modifications are linked to its role as an RNA-binding stress-responsive protein. Based on these studies, we hypothesize a role for nucleolin phosphorylation in regulating mRNA stability of different target genes during DDR. To test this we have engineered a novel system with tet-off promoter in human osteosarcoma cells to express 3xFlag-tagged nucleolin-wt or phospho-mutant [6/S*A, defective in undergoing phosphorylation at six consensus CK2 sites] that allow us to control expression of nucleolin using doxycycline. Cell proliferation assays with these inducible cells indicated that although nucleolin phosphorylation by CK2 was required for cell proliferation in unstressed cells, a hypo-phosphorylated nucleolin mutant caused higher cell viability upon genotoxic stress. Further, nucleolin phospho-variants differentially elevated p53 protein levels and hypo-phosphorylated nucleolin associated with p53 even under non-stressed conditions. Our co-immunoprecipitation (co-IP) assays also showed that nucleolin associated with poly(A)-specific ribonuclease (PARN), an mRNA decay enzyme that controls mRNA stability of different genes during DDR. Importantly, hypo-phosphorylated nucleolin mutant decreased PARN deadenylase activity in vitro. Interestingly, nucleolin also interacted with cellular p53-mRNA as detected by RNA immunoprecipitation assays with nucleolin antibodies followed by qRT-PCR. Our data for the first time provides evidential support that nucleolin phosphorylation can play a regulatory role in driving the cell cycle and controlling gene expression during DDR. Direct associations of nucleolin with p53 and PARN probably affect p53-signaling as well as PARN-deadenylase activity. Understanding the control of gene expression that is regulated by nucleolin phosphorylation during DDR, will provide valuable insights into the mechanism(s) behind the cellular decisions of cell survival or death upon DNA damage. Citation Format: Xiaokan Zhang, Shu Xiao, Emral Devany, Zaineb Nadeem, Elif Caglar ýþýn, Frida Kleiman, Anjana D. Saxena. Nucleolin phosphorylation mediated regulation of gene expression in determining cellular fate during the DNA damage response (DDR). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3188. doi:10.1158/1538-7445.AM2013-3188</jats:p