Sbp1 modulates the translation of Pab1 mRNA in a poly(A)- and RGG-dependent manner

Methylation of RGG domain disrupts protein–protein but not protein–RNA interactions. (A) GST-pulldown assay showing RGG methylation in Sbp1 (Sbp1^m) disrupts the Sbp1–Pab1 interaction. Sbp1^m was incubated with GST-Pab1 without 5′UTR_Pab1 (lane 2), in the presence of 10× 5′UTR_Pab1 (lane 3) or RNase A (lane 4). The protein was incubated with GST as a negative control (lane 1). (B) IgG-pulldown assay showing Sbp1^m compromises ternary complex formation. Sbp1^m and Pab1 were incubated with eIF4G1-TAP without 5′UTR_Pab1 (lane 2), in the presence of 10× 5′UTR_Pab1 (lane 3), or an incubation followed by RNase A treatment (lane 4). Sbp1^m and eIF4G1 show no interaction in the presence of 5′UTR_Pab1 (lane 1). (C) EMSA showing that methylation of RGG repeats in Sbp1 does not affect RNA binding. ³²P-labeled 5′UTR_Pab1 was incubated with 10× Sbp1 (lane 2) or 10× methylated Sbp1 (lane 3). Lane 1 is 5′UTR_Pab1 alone.