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Liu, Rui Wen

Protein arginine methylation is a post-translational modification critical to various biological processes, including epigenetic regulation, RNA processing, and signal transduction. A family of enzymes known as protein arginine N-methyltransferases (PRMTs) are responsible for catalyzing the transfer of methyl groups to arginine residues of their target proteins, such as histones, nucleosomes, transcription factors, and various heterogeneous nuclear ribonucleoproteins (hnRNPs). The prevalence of PRMTs in various biological pathways has made them promising druggable targets for various diseases like neurodegenerative diseases and cancer. PRMT2, in particular, is associated with various cancers, including glioblastoma (GBM), breast cancer, hepatocellular carcinoma (HCC), and renal cell carcinoma (RCC). However, its low enzymatic activity has posed a challenge in characterizing its functions and further elucidating its roles in the disease-causing pathways. This project aims to validate and develop a high-throughput phenotypic assay in a yeast model system to screen for PRMT activities. Previous research has generated a temperature-sensitive mutant of Npl3p, a yeast hnRNP protein involved in mRNA export from the nucleus. Yeast carrying this npl3-1 genetic mutation are unable to grow at temperatures above 34˚C due to aberrant nucleocytoplasmic localization that results in cell lethality at elevated growth temperatures. It has also been discovered that the overexpression of yeast Hmt1p and its human homologue, PRMT1, could functionally rescue this temperature-sensitive growth phenotype, while PRMT2 could not. In this project, the yeast strain PSY773, carrying the npl3-1 mutation, was transformed with genes encoding human PRMT1 and PRMT2 using two pGAL vectors. Unfortunately, unexpected growth patterns were observed from the transformants grown at the non-permissive temperature. Our troubleshooting efforts concluded that this observation could be attributed to the loss of the npl3-1 allele in the PSY773 stock we obtained, and further experimentation is needed to restore the temperature sensitivity of PSY773 for this assay. Moreover, we also observed a temperature-independent lethality in the yeast when the GFP-Npl3p (A254V) mutant fusion protein was overexpressed, which further implicated the role of Npl3p in yeast viability. The final outcomes of this yeast-based assay could provide us with a unique perspective in further studying the structure and functions of PRMT2 and its variants, contributing to a broader understanding of PRMT2’s biological roles and its linkage to oncogenic pathways.

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University of British Columbia. PHAR 491

Vancouver : University of British Columbia Library

10.14288/1.0442122

Pharmaceutical Sciences, Faculty of

Undergraduate

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