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

Sbp1 binds to 5′UTR_Pab1 specifically and cooperatively. (A) Schematic representation of Sbp1's domain organization. Amino acid residue numbers at the boundaries of individual domains are indicated. (B) An EMSA showing the formation of 5′UTR_Pab1•Sbp1 complex at 1:1 stoichiometry. 5′UTR_Pab1 was incubated with increasing concentration of purified Sbp1 (1:0, 1:5, 1:10, 1:15, and 1:20 of 5′UTR_Pab1:Sbp1 for lanes 1–5, respectively). (C) Competition EMSA showing specific interactions between 5′UTR_Pab1 and Sbp1. Sbp1 was first incubated with ³²P-5′UTR_Pab1 (lanes 2 and 6). An excess of unlabeled 5′UTR_Pab1 by five- and 10-fold (lanes 3 and 4) and its complementary RNA, 5′UTR_{Pab1-complement}, (lanes 7 and 8) were used to compete with ³²P-5′UTR_Pab1 bound to Sbp1. (D) An EMSA showing weak interactions between the 5′UTR_Pab1 and individual domains of Sbp1. Sbp1_RRM1 (lane 2), Sbp1_RGG (lane 4), and Sbp1_RRM2 (lane 6) were incubated with 5′UTR_Pab1. A molar ratio of 1:20 (5′UTR_Pab1:Sbp1 domain mutants) was used in the experiment. 5′UTR_Pab1 alone was shown in lanes 1, 3, and 5.