Thus far, we demonstrated that Sbp1 interacts with Pab1 via its RGG domain in an RNA-dependent manner. It is known that the RRM2 domain of Pab1 binds to eIF4G1 directly (Kessler and Sachs 1998; Safaee et al. 2012). Furthermore, earlier mass spectrometry results show that Sbp1, Pab1, and eIF4G interact with each other (Gavin et al. 2002, 2006). To test formation of a stable 5′UTRPab1–Sbp1–Pab1–eIF4G1 RBP in vitro, we did an IgG-pulldown assay using purified eIF4G1, Pab1, and Sbp1. In this experiment, in order to ensure the correct folding and functionality of the protein, eIF4G1 was chromosomally expressed and affinity-purified in yeast. Furthermore, endogenous RNAs that bind to eIF4G1 and Pab1 were removed (see Materials and Methods) to minimize interference of the bound RNAs to protein–protein interactions. As shown in Figure 4 (cf. lanes 2 and 3), the three proteins bind simultaneously only in the presence of 5′UTRPab1. As expected, formation of the ternary complex (Sbp1–Pab1–eIF4G1) on the 5′UTRPab1 was disrupted by the addition of RNase A (Fig. 4, cf. lanes 3 and 4), suggesting the importance of the RNA in the ternary complex formation.