Pab1 in yeast is a 71 kDa RNA-binding protein with four RNA recognition motifs (RRM1–4) and a C-terminal protein-binding domain, also known as PABC (Wigington et al. 2014). To determine which regions in the Pab1 interact with Sbp1, we generated domain mutants of Pab1 and tested their bindings with Sbp1. Our results show that the protein construct containing the RRM1–4 domains of Pab1, Pab1RRM1–4, binds to Sbp1 (Fig. 3E). In contrast, no molecular interaction was detected when PABC, or individual RRM domain mutants including Pab1RRM1, Pab1RRM2, Pab1RRM3, and Pab1RRM4 as well as other tandem RRM domains, were tested (Supplemental Fig. S5A–H). These observations, suggest that, together, RRM1–4 domains in Pab1 are responsible for the interaction with Sbp1. Consistent with our previous observations, Sbp1 and the RRM1–4 of Pab1 only interact with each other in the presence of 5′UTRPab1 (Fig. 3E, cf. lanes 2 and 3), and addition of RNase A abolishes the protein–protein interaction (Fig. 3E, cf. lanes 3 and 4). Taken together, these results confirm that molecular interactions between Pab1RRM1-4 and Sbp1 are RNA-dependent: Binding of the RNA is important to expose the RGG repeats of Sbp1, which is required for the interaction with Pab1RRM1–4.