Patient 3’UTR mutations regulate post-transcriptional gene expression and cellular oncogenicity in prostate cancer

Abstract

While cancer genomics has historically focused on the coding genome, the 3' untranslated region (3’UTR) represents a largely unexplored avenue for uncovering alternative genetic drivers of disease. The 3’UTR controls mRNA stability and translation through sequence and structure-based features that recruit trans-acting factors such as RNA-binding proteins (RBPs) and microRNAs (miRNAs). Though many studies have discovered ways in which RBPs, miRNAs, or other aspects of the 3’UTR are oncogenic, little is known about how somatic mutations to the 3’UTR may function in cancer. In this dissertation, I discuss how I built and utilized a pair of massively parallel reporter assays (MPRAs) to better understand the role of 3’UTR mutations in prostate cancer. Using these novel tools, I measured how thousands of 3’UTR mutations found in advanced stage prostate tumors affect multiple levels of post-transcriptional gene expression, uncovering hundreds of patient mutations that significantly affect either mRNA stability or translational efficiency. Additionally, I performed CRISPR-Cas9 base editing to demonstrate that introduction of oncogenic patient 3’UTR mutations into their endogenous genomic context leads to increased cell growth and stress resistance. In parallel, I applied this same base editing technology to a patient mutation of interest in the 5’ untranslated region, proving that single-nucleotide changes to either UTR can have significant effects on mRNA translation. This work represents an unprecedented view of the extent to which disease-relevant UTR mutations affect mRNA stability, translation efficiency, and cancer, where use of cutting-edge technology brings us closer to understanding the full breadth of genetic drivers in cancer.