Sbp1 interacts with Pab1 in an RGG- and RNA-dependent manner. (A–C) GST-pulldown assays showing interactions of GST-tagged Pab1 with Sbp1 (A), Sbp1RGG (B), and Sbp1ΔRGG (C). Sbp1RGG stands for RGG domain of Sbp1; Sbp1ΔRGG stands for mutant with RGG domain replaced by glycine–serine repeats. Purified proteins were incubated in the absence of 5′UTRPab1 (lane 2), with 10× 5′UTRPab1 (lane 3) or with 10× 5′UTRPab1 followed by RNase treatment (lane 4). The proteins pulled down are shown by SDS–PAGE. Lane 1 shows the negative control containing GST only. (D) RNA-binding of Sbp1, not Pab1, is important for the Sbp1–Pab1 interaction. GST-pulldown assays showing Sbp1–Pab1 interaction under different conditions: Sbp1 incubated with Pab1 in the absence of 5′UTRPab1 (lane 2), GST-tagged Pab1 incubated with the Sbp1•5′UTRPab1 complex (lane 3), and Sbp1 incubated with the GST-Pab1•5′UTRPab1 complex (lane 4). In lane 4, 5′UTRPab1 was incubated with GST-Pab1 first, and unbound RNA in excess was removed prior to the addition of Sbp1. Lane 1 shows negative control with GST only. (E) Deletion of the C-terminal domain of Pab1 does not affect Sbp1–Pab1 interaction. GST-pulldown assay was performed for GST-Sbp1 and the C-terminal deleted variant Pab1 (Pab1RRM1-4). Sbp1 and Pab1RRM1-4 interact with each other in the presence of 5′UTRPab1 (cf. lanes 2 and 3), and RNase treatment disrupts the protein interaction (cf. lanes 3 and 4). Proteins were incubated with GST as the negative control (lane 1).