To study roles of the arginine methylation in mediating protein–protein as well as protein–RNA interactions, we first obtained Sbp1 with the methylated RGG domain by coexpressing Sbp1 with its cognate methyltransferase Hmt1 (Supplemental Fig. S2A; Hsieh et al. 2007). We then confirmed methylation of arginines in the RGG domain by mass spectrometry (Supplemental Fig. S6). Next, using the same GST-pulldown of purified proteins and EMSA as described in the previous sections, we demonstrated that RGG methylation (RGGm) compromises the binding of Sbp1 to Pab1 (Fig. 5A, cf. lanes 3 and 4) and to the formation of the ternary complex (Sbp1–Pab1–eIF4G1) on the 5′UTRPab1 (Fig. 5B, cf. lanes 3 and 4). In contrast, methylation of the RGG domain has no influence on the binding of Sbp1 to 5′UTRPab1 (Fig. 5C, cf. lanes 2 and 3).