Arginine Methylation of Yeast mRNA-binding Protein Npl3 Directly Affects Its Function, Nuclear Export, and Intranuclear Protein Interactions

Anne E Mcbride,Kelly A Mcgrath,Elizabeth A Stemmler,Jeffrey T Cook,Jeffrey A Rubens,Kate L Rutledge

doi:10.1074/jbc.m505831200

Abstract

Arginine methylation can affect both nucleocytoplasmic transport and protein-protein interactions of RNA-binding proteins. These effects are seen in cells that lack the yeast hnRNP methyltransferase (HMT1), raising the question of whether effects on specific proteins are direct or indirect. The presence of multiple arginines in individual methylated proteins also raises the question of whether overall methylation or methylation of a subset of arginines affects protein function. We have used the yeast mRNA-binding protein Npl3 to address these questions in vivo. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry was used to identify 17 methylated arginines in Npl3 purified from yeast: whereas 10 Arg-Gly-Gly (RGG) tripeptides were exclusively dimethylated, variable levels of methylation were found for 5 RGG and 2 RG motif arginines. We constructed a set of Npl3 proteins in which subsets of the RGG arginines were mutated to lysine. Expression of these mutant proteins as the sole form of Npl3 specifically affected growth of a strain that requires Hmt1. Although decreased growth generally correlated with increased numbers of Arg-to-Lys mutations, lysine substitutions in the N terminus of the RGG domain showed more severe effects. Npl3 with all 15 RGG arginines mutated to lysine exited the nucleus independent of Hmt1, indicating a direct effect of methylation on Npl3 transport. These mutations also resulted in a decreased, methylation-independent interaction of Npl3 with transcription elongation factor Tho2 and inhibited Npl3 self-association. These results support a model in which arginine methylation facilitates Npl3 export directly by weakening contacts with nuclear proteins.

Highlights

Protein-arginine methylation by type I methyltransferases, which add one or two methyl groups to one of the guanidino nitrogens of arginine, has been shown to affect a number of eukaryotic processes including protein transport, transcription, and cell signaling
We present evidence that overall methylation is important for protein function through the introduction of systematic arginine-to-lysine mutations in RGG tripeptides followed by tests of Npl3 function in a yeast strain that requires Hmt1
Npl3 is a interesting model to explore the effects of arginine methylation on protein function because of its extensive arginine-glycine-rich domain, which has 15 RGG, 3 RG, and 2 RXR peptides (Fig. 1)

Summary

TABLE I Yeast strains used in this study

MAT␣ nup49::TRP1 ade ade ura leu his ϩ pUN100-nup49ts-LEU2 MAT␣ hmt1⌬::HIS3 ade ade ura leu his lys MATa ade ade ura leu his lys MAT␣ npl3⌬::HIS3 ade ade can ura leu his lys trp ϩYCp50-NPL3–3 MATa hmt1⌬::HIS3 nup49::TRP1 ade ade ura leu lys ϩ pUN100-nup49ts-LEU2 MATa npl3⌬::HIS3 hmt1⌬::HIS3 ade ade ura leu his lys ϩ YCp50-NPL3–3 MATa THO2–9xmyc:TRP1 ura leu his lys trp MAT␣ hmt1⌬::HIS3 THO2–9xmyc:TRP1 ura lys MATa cbp80⌬::HIS3 ura ade ade leu his MATa npl3⌬::HIS3 cbp80⌬::HIS3 ade ade ura leu lys trp1ϩYCp50-NPL3–3 MAT␣ hmt1⌬::HIS3 NPL3-myc:URA3 ade ade ura leu his lys MAT␣ hmt1⌬::HIS3 npl3-RK1–15-myc:URA3 ade ade ura leu his lys MATa NPL3-myc:URA3 ade ade ura leu his lys MATa npl3-RK1–15-myc:URA3 ade ade ura leu his lys MATa THO2–9xmyc:TRP1 NPL3:URA3 ura leu his lys trp MATa THO2–9xmyc:TRP1 npl3-RK1–15:URA3 ura leu his lys trp MAT␣ hmt1⌬::HIS3 THO2–9xmyc:TRP1 NPL3:URA3 ura lys MAT␣ hmt1⌬::HIS3 THO2–9xmyc:TRP1 npl3-RK1–15:URA3 ura lys MAT␣ npl3⌬::HIS3 ade ade can ura leu his lys trp ϩ pPS2389 MATa npl3⌬::HIS3 hmt1⌬::HIS3 ade ade ura leu his lys ϩ pPS2389. The interaction of Npl with two nuclear proteins, Npl itself and transcription elongation factor Tho, is decreased by the presence of the arginine methyltransferase (19) These interactions, combined with the recruitment of both Npl and Hmt to actively transcribed genes (18, 19), suggest that Npl and Hmt may be involved in linking transcription and mRNA export. We describe experiments that suggest that arginine methylation has a direct effect on Npl export, its self-association and its interaction with Tho, through the use of a mutant Npl protein with all RGG arginines changed to lysines (Npl3-RK1-15). These data support a model in which methylation directly affects Npl export by weakening contacts with nuclear proteins

EXPERIMENTAL PROCEDURES

TABLE II Plasmids used in this study

TABLE III Oligonucleotides used in this study

Da ppm

RESULTS

DISCUSSION