RNA recognition motif 2 of yeast Pab1p is required for its functional interaction with eukaryotic translation initiation factor 4G

Abstract

The eukaryotic mRNA 3' poly(A) tail and its associated poly(A)-binding protein (Pab1p) are important regulators of gene expression. One role for this complex in the yeast Saccharomyces cerevisiae is in translation initiation through an interaction with a 115-amino-acid region of the translation initiation factor eIF4G. The eIF4G-interacting domain of Pab1p was mapped to its second RNA recognition motif (RRM2) in an in vitro binding assay. Moreover, RRM2 of Pab1p was required for poly(A) tail-dependent translation in yeast extracts. An analysis of a site-directed Pab1p mutation which bound to eIF4G but did not stimulate translation of uncapped, polyadenylated mRNA suggested additional Pab1p-dependent events during translation initiation. These results support the model that the association of RRM2 of yeast Pab1p with eIF4G is a prerequisite for the poly(A) tail to stimulate the translation of mRNA in vitro.

FIG. 1

Construction and purification of Pab1p variants. (A) Schematic diagram of the Pab1p deletion constructs used in this study. The following amino acids were omitted from each of the constructs: Pab1-ΔRRM1p, 15 to 129; Pab1-ΔRRM2p, 132 to 216; Pab1-ΔRRM3p, 218 to 311; Pab1-ΔRRM4p, 316 to 427; Pab1-ΔCtermp, 430 to 584; and Pab1-105p, 218 to 584. WT, wild type. (B) Purified Pab1p variants. An SDS–10% polyacrylamide gel stained with Coomassie brilliant blue is shown. Positions of molecular weight standards are indicated to the right.

FIG. 2

Poly(A) binding by the Pab1p variants. Determination of equilibrium dissociation constants of Pab1p variants for oligo(A)₂₀ by gel mobility shift analysis. Shown are representative autoradiograms for Pab1-ΔCterm (A), Pab1-ΔRRM2 (B), and Pab1-ΔRRM4 (C). The analysis was also performed for each of the Pab1p variants discussed in this study. ³²P-labeled oligo(A)₂₀ was incubated with increasing amounts of the indicated Pab1p variants. The percentage of radiolabeled RNA being shifted was used to calculated the K_d values (see Materials and Methods). These values are reported in Table 2. (D) Binding of Pab1p variants to poly(A)-Sepharose. Eluates from poly(A)-Sepharose resin incubated with the indicated Pab1p variants were resolved by SDS-PAGE and visualized by Coomassie brilliant blue staining. Initial binding concentrations of the Pab1 proteins were 3 μM for the wild type (WT), ΔRRM2, and ΔRRM4 and 6 μM for Pab1-105p and Pab1-106p.

FIG. 3

RRM2 is required for Pab1p binding to eIF4G. Glutathione resins containing the Pab1p-binding region of eIF4G1 (GST-eIF4G1/187-300p) (A) or eIF4G2 (GST-eIF4G2/200-315p) (B) were incubated with poly(A) and the indicated Pab1p variant. Eluates from these resins were then resolved by SDS-PAGE (12% gel) and visualized by Coomassie brilliant blue staining. Initial concentrations for the Pab1p variants in the binding reaction were 1.5 μM except for Pab1-ΔRRM1p and Pab1-105p, which were at 3 μM. Poly(A) was used at a concentration of 58 μM AMP. WT, wild type.

FIG. 4

RRM2 is required for poly(A) tail-dependent translation in vitro. (A) Reconstitution of translation in Pab1p immunoneutralized extracts with the recombinant Pab1p variants and LUCpA mRNA. The percentage of reconstitution, relative to the wild type (WT), achieved by the addition of the indicated Pab1p variants to the immunoneutralized extract is plotted on the y axis. Values given are the averages of multiple experiments with three different extracts. Each protein was tested over a range of concentrations, and the maximal activation value was used to represent the percent reconstitution. On average, a nonneutralized extract gave 150 U of luminescence in the absence of added protein, and a neutralized extract gave 2.2 U of luminescence. (B) Poly(A) tail-dependent translation in extracts containing different Pab1p variants. Extracts from yeast strains harboring the indicated Pab1p were prepared and assayed for the indicated LUC mRNA translation. Values on the y axis represent the ratio of LUCpA translation to capLUC translation. The translation of capLUC mRNA serves as an internal standard to control for variation in the overall translational activity of each extract. (C) Synergistic activation of translation in extracts containing different Pab1p variants. The ratio of the amount of translation of capLUCpA mRNA to the sum of capLUC and LUCpA mRNA translation [capLUCpA/(capLUC + LUCpA)] within the indicated extract is plotted on the y axis. For panels B and C, the plotted ratios represent the average of at least two experiments with each of two independently prepared extracts. Representative luminescence values for capLUC mRNA translation in each extract were as follows: WT, 39.8; ΔRRM1, 20.3; ΔRRM2, 23.4; ΔRRM3, 79.8; ΔRRM4, 29.4; and ΔCterm, 5.8.

FIG. 5

The Pab1-16 protein associates with eIF4G but does not activate poly(A) tail-dependent translation. (A) Pab1-16p associates with eIF4G. Glutathione resin containing the GST-eIF4G2/200-315p fusion protein was incubated with poly(A) and the indicated Pab1 protein. Eluates from these resins were then resolved by SDS-PAGE (10% gel). The Pab1 proteins were visualized by Western analysis with a Pab1p polyclonal antibody. The initial concentration for Pab1-16p in the binding reaction was 3 μM; Pab1-1p and Pab1-ΔRRM2p were at 1.5 μM. Poly(A) was used at a concentration of 58 μM AMP. WT, wild type. (B) Pab1-16p fails to reconstitute Pab1p-dependent activation of LUCpA translation. Immunoneutralized extracts were supplemented with the indicated Pab1p and then assayed for their translation of LUCpA mRNA. (C) Extracts containing Pab1-16p do not support LUCpA mRNA translation. Translation extracts from strains harboring the indicated Pab1 protein were programmed with either LUCpA or capLUC mRNA. The ratio of the translation of these two mRNAs is plotted. (D) Extracts containing Pab1-16p exhibit decreased levels of translational synergy. The ratio of the amount of translation of capLUCpA to the sum of capLUC and LUCpA mRNA translation within the indicated extract is plotted on the y axis. A representative of the values of capLUC mRNA translation in the Pab1-16p extracts used for panels C and D was 15.6. (See the legend to Fig. 4 for details of panels B to D.)