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

Sbp1 binds to the A-rich region in 5′UTR_Pab1. (A) An in-line probing gel image of 5′UTR_Pab1. The 5′UTR_Pab1 in its undigested form (lane 1) was subjected to partial alkaline digestion (lane 2) and RNase T1 treatment in the presence of urea at 37°C (lane 3) and at 55°C (lane 4). Results on in-line probing of 5′UTR_Pab1 (lane 5) are shown. Double-stranded regions (P1, P2, P3, P4, and P5) and poly(A)-rich region in the RNA are shown. (B) An in-line probing gel image of 5′UTR_Pab1 in the presence of Sbp1. 5′UTR_Pab1 RNA partially digested by RNase T1 at 55°C in the presence of urea (lane 1). The cleavage pattern of 5′UTR_Pab1 after the in-line probing without Sbp1 (lane 2) and with Sbp1 (lane 3) showing protection of the A-rich region by Sbp1. (C) An EMSA showing the formation of 5′UTR_Pab1•Sbp1•Pab1 complex. Addition of Sbp1 or Pab1 leads to formation of the RNP complexes of 5′UTR_Pab1•Sbp1 (lanes 2 and 3) or 5′UTR_Pab1•Pab1 (lane 4), respectively, and results in a slower electrophoretic mobility of the ³²P-labeled 5′UTR_Pab1. In the presence of Sbp1 and Pab1 simultaneously, a band that migrates even slower appears, suggesting the formation of 5′UTR_Pab1•Sbp1•Pab1 complex (lanes 5 and 6). Lane 1 shows 5′UTR_Pab1 only.