Controlling p53 Responses in AKI

Background: The guardian of the genome p53 is essential for controlling the cell cycle, genome integrity, and tissue repair. p53 is also an important determinant of AKI outcomes. However, limited strategies are available to control the p53 expression and facilitate tissue recovery. Methods: Multiple models of AKI were used in our study. The full-length cDNA from endotoxin-treated mouse kidneys was sequenced using Nanopore long-read sequencing. CRISPR knock-out strategy was used to produce a cryptic exon knock-out mouse. For in vitro experiments, several antisense nucleotides targeting the short isoform were synthesized and transfected into HEK cells. CRISPR knock-in strategy was also used to generate p53 mutant human cell lines. Results: We identified a previously uncharacterized short transcript consisting of the first p53 canonical exon and a cryptic exon embedded in the first p53 intronic region. We found that this short transcript emerges under various stress conditions. To determine the role of this transcript, we generated a mouse model in which the cryptic exon is genetically removed. We found that the lack of the cryptic exon heightens the expression of the canonical p53 upon stress challenges. Notably, mice lacking the short transcript were protected against ischemia-reperfusion injury with a significant increase in the expression of the canonical p53 in various cell types. Next, we designed and screened splice-switching antisense oligonucleotides. Our screening revealed specific antisense targeting sites that enabled us to differentially control the expression of the short transcript and canonical p53. Specifically, we found that targeting the transcription termination site results in the upregulation of the short transcript, linking transcription control and p53-mediated stress responses. Conclusion: We have identified a strategy to control the expression of p53 in the kidney. This strategy involves targeting the stress-responsive cryptic exon, opening the door for finetuning p53 responses in AKI. Funding: Other NIH Support - R01