Regulation of p21 Function by a Sense Long Non-Coding RNA Generated by Alternative Polyadenylation

<p>As cells are continually undergoing damage to the genome, a coordinated change in gene expression across thousands of genes is required for the progression of the DNA damage response (DDR). After initial sensing of DNA damage, the transcription factor p53 induces expression of target genes to halt the cell-cycle to allow DNA repair and induce either senescence or apoptosis. One of the p53 target genes is <em>CDKN1A</em> which encodes the cyclin dependent kinase inhibitor p21. p21 halts cell-cycle progression in response to DNA damage and can also induce senescence. However, p21 expression in cancer cells undergoing chemotherapy allows survival by entering a senescent state in a process called therapy-induced-senescence, highlighting the complexity of p21 function in cancer therapy. Diverse mechanisms are in place to regulate changes in gene expression during DDR as thousands of genes need to be rapidly up- or down-regulated. While there is a general downregulation in transcription, translation, and degradation of existing transcripts during the DDR, some genes need to be upregulated at different times of the DDR. Regulation of 3’ mRNA processing allows fast control of gene expression transcriptome wide as it is a ubiquitous co-transcriptional processing step. One powerful method widely used to control gene expression is alternative polyadenylation (APA). Approximately 70% of eukaryotic transcripts have multiple possible polyadenylation sites (PAS), allowing expression of mRNA isoforms with different 3’ untranslated regions (3’ UTR) as well as different open reading frames. Interestingly, there is an increase in intronic APA events after DNA damage in genes that are involved in DDR, such as <em>CDKN1A</em>.</p> <p>Intronic APA in <em>CDKN1A</em> results in expression of a long non-coding RNA (lncRNA) composed of non-protein coding exon 1 and an alternative last exon composed of a region of intron 1. We termed this lncRNA <em>SPUD</em> (Selective Polyadenylation Upon Damage). <em>SPUD</em> undergoes splicing, is exported to the cytoplasm, and is expressed under the control of p53. Interestingly, siRNA-mediated depletion of <em>SPUD</em> does not affect <em>CDKN1A</em> full-length mRNA levels, but significantly decreases p21 protein levels, suggesting a translational regulatory role of <em>SPUD</em> over p21. In this dissertation my aims were to:</p> <p>1) Investigate the mechanism of action by which <em>SPUD</em> regulates p21 translation.</p> <p>2) Determine whether <em>SPUD</em> regulates p21-medated cellular functions, such as</p> <p>senescence induction and apoptosis inhibition.</p> <p>In Chapter II, I determine that <em>SPUD</em> not only regulates p21 translation in non-stressed cells but also during the DDR. Additionally, I demonstrated that <em>SPUD</em> binds to two RNA binding proteins (RBPs), calreticulin (CRT) and CUGBP1, which were previously described to compete for binding to <em>CDKN1A</em> full-length mRNA and have opposite effects on p21 translation. Importantly, these two RBPs also compete for binding to <em>SPUD</em>. Furthermore, I showed that <em>SPUD</em> binds strongly to CRT, the translational repressor of p21, at times of active p21 translation, suggesting <em>SPUD</em> functions as a molecular decoy to allow p21 translation.</p> <p>In Chapter III, I determine that <em>SPUD</em> depletion does not affect apoptosis induction after DNA damage-inducing doxorubicin treatment commonly used in cancer treatment. However, <em>SPUD</em> depletion does significantly decrease the number of surviving cancer cells that enter senescence. This change in the number of senescent cells is not due to changes in cell viability. Furthermore, <em>SPUD</em> depletion in combination with the senolytic drug navitoclax significantly reduces the number of surviving senescent cancer cells, suggesting that <em>SPUD</em> depletion in combination with DNA damaging/senolytic therapy might be a potential approach to mitigate the effects of surviving therapy-induced senescent cancer cells.</p>

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biology

Grantor

The Graduate School and University Center of The City University of New York

Year dc:date.available

2024

Author dc:creator

• Ramadei, Anthony

Advisor dc:contributor.advisor

• Frida Kleiman

Committee members dc:contributor.committeemember

• Anjana Saxena
• Diana Bratu
• Shahana Mahajan
• Kevin Ryan
• Kristy Brown

• Cancer Biology
• Cell Biology
• Molecular Biology
• Senescence
• Apoptosis
• mRNA Processing
• Cancer

Repository record dc:identifier

https://academicworks.cuny.edu/gc_etds/5936

OAI identifier oai:identifier

oai:academicworks.cuny.edu:gc_etds-7046