Hypoxia alters expression and activity of RNA modifying enzymes targeting cytidine in hepatocellular carcinoma cells

• Main Author: Lee, Benjamin Sian Teck
• Other Authors: Valerie Lin
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2025
• Subjects: Chemistry; Medicine, Health and Life Sciences; Epitranscriptomics; Cancer; Hypoxia; RNA; Epigenetics
• Online Access: https://hdl.handle.net/10356/182898
• Institution: Nanyang Technological University

Cells adapt to environmental changes through a variety of biochemical processes. Some of these adaptations involve introducing chemical modifications to the molecules that make up their genetic material, which are the nucleic acid bases of DNA. While these changes do not alter the underlying DNA sequence, they can influence how active the genes are by affecting the interactions between the DNA and other biomolecules. The bases of RNA can similarly be modified. Termed RNA epigenetics or epitranscriptomics, the mechanisms, processes, and functions of these modifications are a field of active research. Closely related to this is the study of the proteins that install, remove, and recognize these modifications. The objective of this PhD thesis is to study how the levels and activities of these proteins are affected by changes in microenvironmental oxygen concentration using both existing and novel techniques. It will be divided into three main parts. The first part of this thesis tests out various methods of isolating protein-RNA adducts formed by exposure of cultured cells to UV radiation. At the time this project began, the only method available for system-wide characterization of such adducts only targeted proteins bound to mRNA. This was due to its reliance on oligo-d(T) beads for pull-down of the adducts. Since then, however, multiple methods suitable for looking at the protein interactome of total RNA have become available. In my experiments, I have found silica-column based methods to be ineffective at accomplishing this task. On the other hand, methods exploiting phenol-chloroform organic phase separation performed very well, successfully enriching known RNA-binding proteins in UV crosslinked samples. The second part of this thesis uses orthogonal organic phase separation to profile changes in the RNA-binding proteome in hepatocellular carcinoma cells experiencing hypoxic stress. Whole proteome profiling was performed alongside this for comparison. Interestingly, western blot on whole cell lysate revealed the RNA modifying enzyme NAT10 to be downregulated in hypoxic conditions. Targeted epitranscriptomic sequencing experiments on a NAT10 target site on rRNA revealed cytidine acetylation to also be reduced upon hypoxia treatment, raising the question of what other RNA modifications could be affected by hypoxia. The third part of this thesis examines cytidine methylation in the context of hypoxia in hepatocellular carcinoma cells. Since there are multiple enzymes that can catalyse the formation of 5-methylcytidine on RNA, I was interested to know which of these enzymes would be dysregulated under hypoxia as this could potentially lead to a new therapeutic target for hepatocellular carcinoma. Using a novel method which I developed to profile the activity of these enzymes towards total RNA, I found that NSUN2 was more enzymatically active during hypoxia even though no changes in protein abundance was observed. Additionally, the activity of the rRNA methyltransferase NSUN1 was downregulated, which is noteworthy given the findings in the second part of this thesis. Taken together, these findings have shed new insights into the role of RNA modifications in the hypoxia response of hepatocellular carcinoma cells. Future work should strive to examine if inhibition of NSUN2 will have a noticeable effect on cancer survival or progression, and investigate the mechanisms underpinning the downregulation of rRNA modifying enzymes NAT10 and NSUN1 in the hopes of identifying other new druggable targets for hepatocellular carcinoma.