Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation

Abstract

Translation of most eukaryotic mRNAs involves the synergistic action between the 5' cap structure and the 3' poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.

Figure 1.

(A) Schematic diagram, not to scale, of the constructs used in this study. The cap structure in the RNAs is indicated by a black circle, the 100 nt length poly(A) tail is indicated by A(100). (B) Capped/polyadenylated (+/+), capped/non-polyadenylated (+/−), uncapped/polyadenylated (−/+) or uncapped/non-polyadenylated (−/−) Globin-renilla RNAs were translated at 0.05, 0.1, 0.15 or 0.3 mg/ml in the untreated RRL (left panel) or the nuclease-treated lysate (right panel) as described in Materials and Methods section. Translational products were analysed by 15% SDS–PAGE and autoradiography. Synthesis of endogenous globin and lipoxygenase together with exogenous renilla products are indicated by arrows on the figure. Densitometric quantification of renilla synthesis at 0.1 mg/ml from Globin-renilla RNA translation in the untreated and nuclease-treated lysates has been plotted at the bottom of each panel. (C) Capped/polyadenylated (+/+), capped/non-polyadenylated (+/−), uncapped/polyadenylated (−/+) or uncapped/non-polyadenylated (−/−) Globin-renilla RNAs were translated at 0.05, 0.1, 0.15 or 0.3 mg/ml in the untreated RRL (left panel) or the nuclease-treated lysate (right panel) as described in Materials and Methods section. Renilla luciferase activity was then determined and normalized as described in Materials and Methods section. The results presented are representative of three independent experiments are expressed as means ± SD.

Figure 2.

(A) Different combinations of Globin-renilla RNAs (+/+), (+/−), (−/+) or (−/−) were translated at 0.1 mg/ml for 0, 15, 30 or 60 min in the nuclease-treated RRL (left panel) or untreated RRL (right panel) as indicated on the figure. Renilla luciferase activity was then determined and normalized as described in Materials and Methods section.(B) Globin-renilla RNAs (+/+), (+/−), (−/+) or (−/−) at 0.1 mg/ml were translated in both nuclease-treated RRL (left panel) or untreated RRL (right panel) in the presence of 0, 0.25, 0.5 or 1 mM added MgCl₂. The resulting translation products were resolved by 15% SDS–PAGE and autoradiography and the position of the protein products are indicated by arrows on the figure. (C) Globin-renilla RNAs (+/+), (+/−), (−/+) or (−/−) at 0.1 mg/ml were translated in both nuclease-treated RRL (left panel) or untreated RRL (right panel) in the presence of 0, 0.25, 0.5 or 1 mM added MgCl₂. Renilla luciferase activity was then determined and normalized as described in Materials and Methods section. The results presented are representative of three independent experiments are expressed as means ± SD.

Figure 3.

(A) Capped and polyadenylated Globin-renilla RNAs (+/+) and uncapped and non-polyadenylated HCV-renilla RNAs (−/−) were translated in the untreated reticulocyte lysate that had been pre-incubated with 50, 100 or 250 ng of GST-Paip2 recombinant protein or (B) 10, 20 or 30 ng of free poly(rA). Protein production was determined by densitometric quantification of the renilla protein product and is expressed as percent of the control (no inhibitor added, set to 100%). Data were obtained from densitometric analyses of at least three independent experiments and are expressed as means ± SD.

Figure 4.

(A) Uncapped polyadenylated (−/+) and uncapped non-polyadenylated (−/−) EMCV-renilla RNAs were translated at 0.5 mg/ml during 30 min at 30°C in both the nuclease-treated (left panel) and untreated RRLs (right panel) that were pre-incubated with 0, 0.2, 0.4 or 0.6 µl of an in vitro-synthesized FMDV L protease (see Materials and Methods section). Translation products were resolved by 15% SDS-PAGE followed by autoradiography. Densitometric analysis of the renilla protein product was performed and expressed as percentage of the control (100%, no l protease added) at the bottom of each panel. (B) At the end of the in vitro translation incubation, samples (1 µl) from the experiment described above were run on a 10% SDS–PAGE and the proteins transferred to PVDF membrane and incubated with antibodies specific to the C-terminal part of eIF4GI.

Figure 5.

Different combinations of Globin-renilla RNAs (+/+), (+/−), (−/+) or (−/−) were translated at 0.1 mg/ml for 30 min at 30°C in the nuclease-treated RRL (left graph) or nuclease-treated RRL supplemented with 0.15 mg/ml of globin mRNAs (Gibco) (right graph) as indicated on the figure. Translation products were quantified by analysis of luciferase activity.