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

Author

Anthony Ramadei, The Graduate Center, City University of New YorkFollow

Date of Degree

9-2024

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biology

Advisor

Frida Kleiman

Committee Members

Anjana Saxena

Diana Bratu

Shahana Mahajan

Kevin Ryan

Kristy Brown

Subject Categories

Cancer Biology | Cell Biology | Molecular Biology

Keywords

Senescence, Apoptosis, mRNA Processing, Cancer

Abstract

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 CDKN1A 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 CDKN1A.

Intronic APA in CDKN1A 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 SPUD (Selective Polyadenylation Upon Damage). SPUD undergoes splicing, is exported to the cytoplasm, and is expressed under the control of p53. Interestingly, siRNA-mediated depletion of SPUD does not affect CDKN1A full-length mRNA levels, but significantly decreases p21 protein levels, suggesting a translational regulatory role of SPUD over p21. In this dissertation my aims were to:

1) Investigate the mechanism of action by which SPUD regulates p21 translation.

2) Determine whether SPUD regulates p21-medated cellular functions, such as

senescence induction and apoptosis inhibition.

In Chapter II, I determine that SPUD not only regulates p21 translation in non-stressed cells but also during the DDR. Additionally, I demonstrated that SPUD binds to two RNA binding proteins (RBPs), calreticulin (CRT) and CUGBP1, which were previously described to compete for binding to CDKN1A full-length mRNA and have opposite effects on p21 translation. Importantly, these two RBPs also compete for binding to SPUD. Furthermore, I showed that SPUD binds strongly to CRT, the translational repressor of p21, at times of active p21 translation, suggesting SPUD functions as a molecular decoy to allow p21 translation.

In Chapter III, I determine that SPUD depletion does not affect apoptosis induction after DNA damage-inducing doxorubicin treatment commonly used in cancer treatment. However, SPUD 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, SPUD depletion in combination with the senolytic drug navitoclax significantly reduces the number of surviving senescent cancer cells, suggesting that SPUD depletion in combination with DNA damaging/senolytic therapy might be a potential approach to mitigate the effects of surviving therapy-induced senescent cancer cells.

Recommended Citation

Ramadei, Anthony, "Regulation of p21 Function by a Sense Long Non-Coding RNA Generated by Alternative Polyadenylation" (2024). CUNY Academic Works.
https://academicworks.cuny.edu/gc_etds/5936