Sbp1 inhibits both cap-dependent and cap-independent translation initiation of Pab1 mRNA in an RGG- and RNA-dependent manner. Reporter constructs are shown schematically in the top panel of the figure. In vitro cap-dependent and cap-independent translation activities of 5′UTRPab1 and 5′UTRPab1-Δpoly(A) in the presence of different concentrations of Sbp1 and its variants are shown. (A) Changes of cap-dependent translation initiation activities of 5′UTRPab1 by Sbp1 and its protein variants. In vitro translation assays were performed by incubating the capped 5′UTRPab1 followed by firefly luciferase gene and poly(A) tail [5′UTRPab1-FLuc-poly(A)] in ΔSbp1 cell extract with varying amounts of Sbp1, Sbp1ΔRGG, Sbp1m, Sbp1RGG, and Sbp1mRGG added. Sbp1ΔRGG: a substitution of RGG domain with glycine–serine repeats; Sbp1m: methylated Sbp1; Sbp1RGG: RGG domain of Sbp1; Sbp1mRGG: methylated RGG domain. (B) Changes of cap-dependent translation initiation activities of 5′UTRPab1 at varying concentrations of Sbp1 added to WT cell extracts. (C) Changes of cap-independent translation initiation activities of 5′UTRPab1 by Sbp1 and its protein variants. Experiments were done as in A using a different reporter construct, the uncapped 5′UTRPab1-FLuc-poly(A), which has a hairpin insertion before the 5′UTRPab1 to inhibit ribosome scanning. (D) Changes of cap-dependent translation initiation activities of 5′UTR4E by Sbp1 and its protein variants. Experiments were done as in A, except the capped 5′UTReIF4E-FLuc-poly(A) was used. (E) Changes of cap-independent translation initiation activities of 5′UTRCrPV by Sbp1 and its protein variants. Experiments were done as in A, except the uncapped Hairpin-5′UTRCrPV-FLuc-poly(A) was used. (F) Changes of cap-dependent translation initiation activities of 5′UTRPab1-Δpoly(A) by Sbp1 and its protein variants. 5′UTRPab1-Δpoly(A): 5′UTRPab1 RNA with the internal A-rich region deleted. The integrity of the RNAs was confirmed by either northern blot or ethidium bromide-stained gels under denaturing conditions. Luciferase activities are normalized to the amount of capped or uncapped RNAs and the total protein levels in the study. Reported translation activities are the average of results obtained in three independent experiments. Controls including translation activities of the blank, 5′UTR of eIF4E (5′UTReIF4E) and internal ribosome entry site of cricket paralysis virus (5′UTRCrPV) under the same experimental conditions are shown in D and E.