| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | Alba3 hypomethylated mutants were generated by replacing arginine residues (RG/RGG motifs) in the C-terminus to either tryptophan (WGG) or lysine (R192K or KGG) and inserting each construct into pFLAG_HA-Alba3. For the latter, alba3sequence was modified by Q5® Site-Directed Mutagenesis (New England Biolabs), according to manufacturer instructions, using oligos shown in Table S3 to either replace a single arginine detected in the MMA methyl-SILAC analysis (Alba3R192K) or all the eleven arginine residues in RGG motifs present in its C-terminal tail (Alba3KGG). The Alba3WGG mutant sequence was obtained as a custom synthetic gene (GenScript), in which each of the eleven arginine residues were mutated. Final constructs were transfected into both WT and Δprmt7 L. major cell lines. | Get A Quote |
RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine MethylTransferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypom... More
