RUI: Protein Arginine Methylation and Nucleocytoplasmic Transport

Bowdoin College, Brunswick ME

Investigators

Abstract

The transport of messenger RNAs from the site of transcription in the nucleus to the site of translation in the cytoplasm requires the coordinated efforts of an eclectic array of RNA-binding proteins and transport factors. The methylation of arginine-glycine (RG)-rich RNA-binding proteins, which have been implicated in transport and other steps of RNA metabolism, suggests that this post-translational modification may play a role in formation, movement, or dissociation of ribonucleoprotein (RNP) complexes. Protein arginine methylation may parallel other processes such as phosphorylation in its complexity and multifunctional roles in the eukaryotic cell, yet to date there is only a rudimentary understanding of how methylation affects the molecular interactions of RG-rich proteins and their function in vivo. The predominant protein arginine methyltransferase, which is conserved from yeast to human, frequently targets a subset of RG-rich proteins that contain numerous RGG tripeptides. There are two possible ways methylation may affect the function of these proteins. First,the overall methylation state of the RGG domain may influence protein function. Second, methylation of specific arginine residues within the RGG domain may be critical for modulation of protein function. The first objective of this work is to distinguish between these two hypotheses by defining in vivo methylation sites in a model RGG protein and by testing the importance of the targeted arginine residues for protein function. The second objective is to determine the role of arginine residues and their methylation in modulating RNP complexes involved in nucleocytoplasmic transport. The availability of facile genetic, cell biological and biochemical assays for the yeast Saccharomyces cerevisiae makes this model eukaryote an excellent system with which to address these objectives. A major mRNA-binding protein involved in mRNA export, Npl3 (nuclear protein localization 3), contains numerous RGG motifs and its nuclear export is facilitated by methylation. Although the molecular mechanism for this effect is unknown, preliminary data suggest that methylation of Npl3 modulates its self-association and its interaction with a protein involved in transcription elongation,Tho2. To distinguish between roles of individual methylarginine residues and overall Npl3 methylation,initially in vivo sites of Npl3 methylation will be determined by matrix-assisted laser desorption/ionization mass pectrometry. Site-directed mutagenesis of methylated arginine residues combined with genetic tests for mutant Npl3 function will be used to ascertain the importance of these residues and their methylation in the cell. Molecular effects on protein function will then be probed by monitoring mutant Npl3 self-association,nuclear export, and interaction with the Tho2 protein. In addition, an assay will be developed to test the effects of self-association on Npl3 export. Finally, analysis of genetic interactions between NPL3 and genes involved in transcription elongation will offer insight into the functional importance of the Npl3-Tho2 interaction. These experiments, which combine detailed molecular analysis with in vivo tests for function, will lead to a greater understanding of the impact of arginine residues and their methylation on dynamic complexes in the eukaryotic cell. The recent explosion in the number of putative arginine methyltransferases and the identification of a plethora of potential substrates has generated extensive interest in their mechanisms of action . The conservation of arginine methyltransferases from yeast to human and the ubiquity of RG-rich proteins suggest that the results of this study will lend insight into the significance of arginine methylation in RNP complexes and their function in many other eukaryotic systems. The work will be performed at Bowdoin College as a RUI (Research at Undergraduate Institutions) project. Two undergraduate students will be supported each summer while they engage in research projects in the laboratory. In addition, students will present their work at major national scientific meetings such as the Yeast Genetics and Molecular Biology meeting and/or the American Society for Microbiology meeting.