Specific arginine methylation of Rev by PRMT6 in vitro. A, Sequences of recombinant histidine-tagged Tat86 and Rev. Both sequences are chimeric and consist of BH10 (amino acids 2–66 and 2–15, respectively) and HXB2 (amino acids 67–86 and 16–116, respectively). Underscored are the cysteine rich motif and the ARM of Tat86, as well as the two α-helices of the helix-loop-helix motif of Rev. Arginine residues located in the N-terminal portion of the ARMs are shaded in black. B, Arginine methylation of Rev by PRMT6. Recombinant histidine-tagged Rev was incubated with [methyl-3H]-S-adenosyl-L-methionine in the presence (lane 1) or absence (lane 2) of PRMT6. As a positive control, recombinant histidine-tagged Tat86 was incubated with (lane 6) or without (lane 7) PRMT6. As negative controls, BSA was incubated in the presence (lane 5) or absence (lane 4) of PRMT6, or PRMT6 alone was used (lane 3). Proteins were separated by SDS-PAGE, stained with Coomassie blue (upper panel), and tritium incorporation was screened by fluorography (lower panel). The migratory positions are indicated by arrows on the left. C, Specific arginine methylation of the N-terminal portion of the ARM of Rev by PRMT6. Recombinant histidine-tagged wild-type (lane 1) and mutant Rev proteins (lanes 2–9), as well as BSA (lane 10) as a negative control, were treated as described in B. The Coomassie blue stained gel (center panel) and the developed film (upper panel) were used to calculate the percentages of methylation of the individual mutants (lower panel). The migratory positions are indicated by arrows on the left. Similar results were observed in three experiments. D, AMI1 inhibits arginine methylation of Rev by PRMT6. Recombinant histidine-tagged Rev was incubated with PRMT6, as described in B, in the presence of increasing amounts of AMI1. Band intensities were quantified to calculate the IC50.