UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Insights into a heteromeric protein arginine N-methyltransferase complex Pak, Laam

Abstract

Protein arginine N-methyltransferases (PRMTs) act in signaling pathways and gene expression by methylating arginine residues within target proteins. PRMT1 is responsible for most cellular arginine methylation activity and can work independently or in collaboration with other PRMTs. In this Ph.D. thesis I demonstrated an interaction between PRMT1 and -2 using co-immunoprecipitation and bimolecular fluorescence complementation (BiFC). As a result of this interaction, PRMT2 stimulated PRMT1 methyltransferase activity, affecting its apparent Vmax and Km values in vitro, and increasing the production of methylarginines in cells. Active site mutations and regional deletions on PRMT1 and -2 were also investigated, which demonstrated that complex formation required full-length, active PRMT1. However, the interaction between PRMT1 and -2 proved insensitive to methylation inhibition in the absence of the PRMT2 Src homology 3 (SH3) domain, which suggests that the PRMT2 SH3 domain may mediate this interaction between PRMT1 and -2 in a methylation-dependent fashion. The role of the PRMT2 SH3 domain was investigated through screening for its associated proteins using GST-pull down assays followed by LC-MS/MS proteomic analysis. The result of this study revealed associations of the PRMT2 SH3 domain with at least 29 splicing-related proteins, suggesting a potential role of PRMT2 in regulating pre-mRNA processing and splicing. The interaction between PRMT2 and the Src substrate associated in mitosis of 68 kDa (Sam68) possibly through the PRMT2 SH3 domain was demonstrated using co-immunoprecipitation. Additionally, immunofluorescence results present herein imply that the PRMT2 SH3 domain could affect Sam68 sub-cellular localization in hypomethylated HeLa cells. The biological functions of PRMT2 and the PRMT1/2 heteromeric complex were explored by pursuing the identity of associated proteins common to both PRMT1 and -2 using mass spectrometry proteomics. Approximately 50% of the identified protein hits have reported roles in controlling gene expression, while other hits are involved in diverse cellular processes such as protein folding, degradation, and metabolism. Importantly, three novel PRMT2 binders, p53, promyelocytic leukemia protein (PML), and extra eleven nineteen (EEN) were uncovered, suggesting that PRMT2 could participate in regulation of transcription and apoptosis through PRMT2-protein interactions.

Item Media

Item Citations and Data

License

Attribution-NonCommercial-NoDerivatives 4.0 International

Usage Statistics