Global proteomics studies in our lab reveal two arginine residues on ZFP3 that are methylated; targeted studies later identified four additional sites. To examine the role of ZFP3 and its methylation on the transcriptome regulation, we created T.brucei cell lines that overexpressed either wild type (WT) ZFP3 or one of two methylmutants at the first two identified sites: a methylmimic (R to F) or a hypomethylated mutant (R to K). We then investigated changes in the transriptome by RNAseq of biological replicate samples. When WT ZFP3 was overexpressed 440 genes changes significantly, and GO term analysis highlighted a potential role for ZFP3 in cell cycle control. In contrast, overexpression of either methylmutant resulted in almost no transcriptome changes, indicating the presence of an arginine at the at the methylation sites is necessary for transcriptome regulation. 

We next aimed to understand the role of arginine methylation by modulating the enzyme(s) that modify ZFP3. T.brucei has four protein arginine methyltransferases (PRMTs), and we hypothesize that different PRMTs modify distinct arginine residues on ZFP3. To test this, we preformed in vitro methylation assays using recombinant ZFP3 proteins with R to K mutations at distinct sites and a battery of recombinant PRMTs. Our data shows that ZFP3 is a substrate for all four PRMTs, and that specific PRMTs methylate specific arginines. These findings will now allow us to examine the role of ZFP3 arginine methylation in vivo in cells depleted of specific PRMTs.