Figure 4

PRMT6 reduces the interaction between a Tat-Rev fusion protein and a TAR-RREIIB hybrid. A, Sequences of TAR, TAR-RREIIB hybrid and Tat-Rev fusion protein. In TAR-RREIIB, the TAR bulge was replaced by the RREIIB stem-loop (bold). The Tat-Rev fusion protein contains the first 49 amino acids of Tat and is linked to residues 34–47 of Rev (bold) by means of four alanine residues (underscored). Arginine residues changed by mutagenesis are shaded in black. B, Knock-down of PRMT6 by pSUPER.retro vector expressing PRMT6-siRNA. HeLa cells expressing PRMT6-siRNA were established using the pSUPER.retro-PRMT6 retroviral vector. Cell lysates were separated by SDS-PAGE and immunoblots were performed. The bands corresponding to PRMT6 protein and the control β-actin are indicated by arrows. C, PRMT6 reduces CAT expression due to diminished Rev-RRE interaction. HeLa cells stably transfected with mock siRNA (m, lanes 1–10) or PRMT6-siRNA (P6si, lanes 11–16) were co-transfected with plasmids expressing Tat-Rev (lanes 2,4,6,8,10,11,13,15), pHIV-LTR-RREIIB-CAT (lanes 1–16) and various amounts of myc-tagged PRMT6 (wild-type lanes 7–14, mutant lanes 3–6). At 48 hours post-transfection, CAT assays were performed, separated by TLC and exposed (upper panel). Fold activations, i.e. results of samples (mono-acylated species per total amount of chloramphenicol) divided by those of negative controls without Rev, were calculated from quantified bands (lower panel). The migratory positions are indicated by arrows. Similar results were observed in each of three separate assays. D, Mutant R38K is less susceptible to PRMT6 methyltransferase activity. Wild-type (lanes 2–4) and mutated Tat-Rev fusion proteins (R35K lanes 8–10, R38K lanes 5–7 and R39K lanes 11–13) were co-transfected with variable amounts of wild-type PRMT6 into HeLa cells as described in C (upper panel). The migratory positions are indicated by arrows. Fold activations were calculated as described in C (lower panel). Similar results were obtained in each of three experiments.