Figure 3

PRMT6 methylates Rev and attenuates Rev protein levels in vivo. A, Interaction of Rev and PRMT6. T-REx™-293 cells were transfected with histidine-tagged Rev (lanes 1,2,3,6,7) and myc epitope-tagged PRMT6 (lanes 1,2,4,6,7). Co-IP was carried out with an anti-histidine-tag antibody coupled gel (lanes 2–5,7) and a control gel (lanes 1,6). Eluates were separated by SDS-PAGE, immunoblotted with anti-myc-epitope (lanes 1–5) or anti-histidine-tag antibodies (lanes 6,7) and signals detected with a secondary antibody coupled to HRP. The migratory positions are indicated by arrows on the left. Bottom line: +: antibody coupled gel; -: control gel. B, PRMT6 methylates and attenuates Rev in vivo. HeLa cells were transfected with histidine-tagged Rev (lanes 4–6) and/or wild-type (lanes 3,6) or mutant (lanes 2,5) myc epitope-tagged PRMT6, or no plasmids (lane 1). After 3 hours pulse labeling, cell lysates were separated by SDS-PAGE, Coomassie stained (left panel) and fluorographed (center panel). Cell lysates were also immunoblotted with anti-Rev antibody and detected as described in A (right panel). Loaded amounts of cell lysates are given in μl and the migratory positions are indicated by arrows. C, Rev protein levels are not affected by PRMT6 pre-translationally. HeLa cells were transfected as described in B. Additionally, HeLa cells expressing siRNA against PRMT6 were used. RNA was isolated for reverse transcription and mean Rev amounts determined by rt-RT-PCR were normalized to GAPDH (left panel) or total RNA (right panel). Rev levels were calculated per amount of Rev in Rev only transfected cells and expressed as percentages. The bars represent standard deviations of the mean of three independent experiments, each of which was carried out in duplicates.