Date of Graduation
Epigenetics and Molecular Carcinogenesis
Doctor of Philosophy (PhD)
Mark Bedford, Ph.D.
Taiping Chen, Ph.D.
Sharon Y.R. Dent, Ph.D.
Rick A. Finch, Ph.D.
David G. Johnson, Ph.D.
PRMT5 is a member of a group of proteins that mediate arginine methylation. It is involved in diverse cellular processes, including cell differentiation, splicing, transcription elongation and epigenetic silencing, and its expression is dysregulated in many cancers. Due to its pleiotropic functions, PRMT5 is subject to multi-level regulation. Post-translational modification (PTM) of proteins can modulate an array of cellular processes by regulating both protein interactions and protein structural changes. PRMT5 is commonly found associated with other proteins; these interactions seem to control both its catalytic activity and its substrate specificity. Recently, it became clear that PRMT5 is phosphorylated at a number of residues, which prompted us to investigate whether phosphorylation of PRMT5 regulates its subcellular localization and/or substrate choice, by facilitating phospho-dependent protein-protein interactions.
To study how phosphorylation affects PRMT5 function, protein microarrays were used to identify novel phosphodependent-interacting proteins. This analysis revealed that phosphorylation mediates the interaction of PRMT5 with several SH2-domain containing proteins, 14-3-3 proteins and the FHA domain of MDC1. These novel phospho-dependent PRMT5 interactions suggest that crosstalk between kinases and arginine methyltransferases may play a pivotal role in modulating the different cellular functions of PRMT5.
Additionally, we have found that the C-terminal region of PRMT5 has a recognition motif shared by PDZ domains and 14-3-3 proteins. In order to bind to this motif, 14-3-3 proteins require the C-terminus to be phosphorylated, while PDZ domain recognition is phospho-independent. From these data, a new regulatory mechanism that affects PRMT5 behavior was proposed; the action of kinases and phosphatases on PRMT5 may function as a switch to regulate interactions between 14-3-3 and PDZ domain-containing proteins. We additionally observed this paradigm with a number of proteins, suggesting that this phosphorylation dependent switch, regulating binding to 14-3-3 and PDZ domains, occurs in a wide range of protein-protein interactions.
Among the recently discovered PDZ-binding partners, we have found that PRMT5 interacts with NHERF2, a membrane-associated protein that regulates the sodium ion exchanger NHE3. Through this interaction, PRMT5 is placed in close proximity to the membrane and therefore may regulate the influx of ions through selective ion channels. Overall, we hypothesize that the direct interaction of PRMT5 with selected partners mediates the appropriate localization of PRMT5, allowing it to methylate specific substrates.
Physiological processes including muscle contraction, cell homeostasis and neurotransmission are controlled by the selective conduction of ions across cell membranes. Ion channels and exchangers are proteins that span cell membranes and form channels or pores, facilitating the movement of ions in and out of cells. Abnormal cellular response to the microenvironment is one of the key factors in the progression of many diseases. To study the role of the C-terminus of PRMT5 in vivo, we used CRISPR/Cas9 technology to create a mouse with the last six amino acids of PRMT5 replaced with an HA tag, as well with an altered PRMT5 C-terminus. Both mouse models lack the critical 14-3-3/PDZ binding motif. This approach revealed that the C-terminus of PRMT5 is essential for viability as no homozygous mutant embryos were recovered. Likewise, no homozygous PRMT5Δ+HA ES cell lines could be created. The results described here represent progress toward understanding PRMT5 function and regulation.
PRMT5, phosphorylation, interactions, domain, protein microarray, SH2, PDZ, 14-3-3, FHA