Exploring the roles of arginine methylation and deimination of the multifunctional nuclear proteins PSF and p54nrb

Many proteins undergo chemical modifications that can alter a particular function of protein. A common modification for example includes phosphorylation. Arginine methylation is now emerging as a mainstream post translational modification of proteins, similar to protein phosphorylation. It has recently been shown that the methylation of arginines can be antagonised enzymatically and it is therefore dynamic in nature. This offers exciting prospects that arginine methylation, as a eukaryotic post translational modification, may mirror phosphorylation in its level of complexity. Many RNA binding proteins undergo arginine methylation as a post translational modification. The methylation of arginine residues is thought to structurally alter the protein and therefore perturb protein-protein or protein nucleic interactions. We have recently used novel enrichment procedures to purify a multi protein nuclear complex from mammalian nuclear cell extracts in which PSF (polypyrimidine tract-binding protein associated splicing factor) is bound to p54nrb/NonO (Non-POU-domain-containing, octamer-binding protein) and identified sites of arginine methylation on the protein PSF. It is proposed to characterise the sites of arginine methylation of the multi-functional nuclear proteins PSF and p54nrb and further explore the roles of such modifications. The characterisation of post translational modifications (arginine methylation and citrullination) and exploring the roles played by such modifications on the multifunctional nuclear protein complex is an ideal forum for a multi-disciplinary experimental programme, encompassing aspects of biophysical techniques in the analysis of biological systems. The research will be performed within the Systems Biology group in the Department of Chemical and Process Engineering at the University of Sheffield.

Arginine methylation is now emerging as a mainstream post translational modification of proteins, similar to protein phosphorylation. It has recently been shown that the methylation of arginines on histones can be antagonised enzymatically via deimination of arginine to citrulline, and it is therefore dynamic in nature. This offers exciting prospects that arginine methylation, as a eukaryotic post translational modification, may mirror phosphorylation in its level of complexity. Many RNA binding proteins undergo arginine methylation as a post translational modification. The methylation of arginine residues is thought to structurally alter the protein and therefore perturb protein-protein or protein nucleic interactions. We have recently used novel enrichment procedures to purify the multi protein nuclear complex from Hela nuclear cell extracts in which PSF (polypyrimidine tract-binding protein associated splicing factor) is bound to p54nrb/NonO (Non-POU-domain-containing, octamer-binding protein) and identified sites of arginine methylation in the protein PSF. It is proposed to characterise the sites of arginine methylation and citrullination of the multi-functional nuclear proteins PSF and p54nrb and further explore the roles of such modifications. It is proposed to overexpress, in E.coli, the human PSF and p54nrb proteins. Following overexpression and purification of the PSF and p54nrb proteins it is proposed to analyse the sites of arginine methylation and citrullination, following incubation of the proteins with arginine methyltrasnferases and pepidyl arginine deiminases in vitro. Mass spectrometry will be used to identify the sites of the modifications. In addition the sites of arginine methylation and citrullination will be analysed in vivo. The PSF-p54nrb complex will be enriched from Hela nuclear cell extracts using novel open tube capillaries and the sites of such modifications will be analysed using mass spectrometry. To aid the identifications of the sites of such modifications using mass spectrometry, it is proposed to develop a variety of approaches including the use of stable isotopes including 13C SAM and H218O for the in vitro reactions and 13CD3 methionine for labeling and quantitation of the sites of methylation in vivo. In an approach to study the roles of the post translational modifications of PSF and p54nrb it is proposed to analyse the kinetics of the interactions of both the modified and unmodified proteins with both protein and nucleic acid substrates using surface plasmon resonance. In addition it is proposed to use proteomic approaches to identify novel interacting partners of PSF and p54nrb, moreover study the effects of arginine methylation and deimination on the interacting proteins associated with PSF and p54nrb.