Methylation of RGG domain disrupts protein–protein but not protein–RNA interactions. (A) GST-pulldown assay showing RGG methylation in Sbp1 (Sbp1m) disrupts the Sbp1–Pab1 interaction. Sbp1m was incubated with GST-Pab1 without 5′UTRPab1 (lane 2), in the presence of 10× 5′UTRPab1 (lane 3) or RNase A (lane 4). The protein was incubated with GST as a negative control (lane 1). (B) IgG-pulldown assay showing Sbp1m compromises ternary complex formation. Sbp1m and Pab1 were incubated with eIF4G1-TAP without 5′UTRPab1 (lane 2), in the presence of 10× 5′UTRPab1 (lane 3), or an incubation followed by RNase A treatment (lane 4). Sbp1m and eIF4G1 show no interaction in the presence of 5′UTRPab1 (lane 1). (C) EMSA showing that methylation of RGG repeats in Sbp1 does not affect RNA binding. 32P-labeled 5′UTRPab1 was incubated with 10× Sbp1 (lane 2) or 10× methylated Sbp1 (lane 3). Lane 1 is 5′UTRPab1 alone.