Biochemical characterisation of cap-poly(A) synergy in rabbit reticulocyte lysates: the eIF4G-PABP interaction increases the functional affinity of eIF4E for the capped mRNA 5'-end

Abstract

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to synergistically stimulate translation initiation in vivo. We recently described mammalian cytoplasmic extracts which, following ultracentrifugation to partially deplete them of ribosomes and associated initiation factors, reproduce cap-poly(A) synergy in vitro. Using these systems, we demonstrate that synergy requires interaction between the poly(A)-binding protein (PABP) and the eukaryotic initiation factor (eIF) 4F holoenzyme complex, which recognises the 5' cap. Here we further characterise the requirements and constraints of cap-poly(A) synergy in reticulocyte lysates by evaluating the effects of different parameters on synergy. The extent of extract depletion and the amounts of different initiation factors in depleted extracts were examined, as well as the effects of varying the concentrations of KCl, MgCl(2) and programming mRNA and of adding a cap analogue. The results presented demonstrate that maximal cap-poly(A) synergy requires: (i) limiting concentrations of ribosome-associated initiation factors; (ii) precise ratios of mRNA to translation machinery (low concentrations of ribosome-associated initiation factors and low, non-saturating mRNA concentrations); (iii) physiological concentrations of added KCl and MgCl(2). Additionally, we show that the eIF4G-PABP interaction on mRNAs which are capped and polyadenylated significantly increases the affinity of eIF4E for the 5' cap.

Figure 1

Cap–poly(A) synergy in ribosome-depleted RRL. (A) Schematic representation of the plasmids used in this work. The Xenopus laevis cyclin B2 and HIV-1 p24 coding regions are shown as open boxes. Numbers below coding regions refer to the first and last amino acids of each reporter gene product. The 5′- and 3′-UTRs are depicted as black and speckled boxes, respectively; the translation initiation codon is underlined. Clones were constructed in duplicate, differing by the presence or absence of an A₅₀ insertion (in parentheses) at the EcoRI site used for linearisation prior to transcription. (B) Standard RRL (left) or ribosome-depleted RRL (middle and right) was programmed with in vitro transcribed RNAs derived from the pB2 or p0p24 plasmids in the form indicated above each lane [final RNA concentrations 6.1 µg/ml for B2 mRNAs and 4 µg/ml (the molar equivalent) for 0p24 mRNAs]. The final concentrations of added KCl and MgCl₂ were 115 and 0.6 mM, respectively. Control reactions were programmed with water (no RNA lane). Translations were processed as described in Materials and Methods. An autoradiograph of the dried 20% polyacrylamide gel is shown. The position of the cyclin B2 or HIV-1p24 translation product is marked. Translation efficiency derived from densitometric quantification is plotted below each lane. Relative stimulation of translation was calculated by comparing the translation efficiency of capped and/or polyadenylated RNA to that of the –/– RNA. Cap–poly(A) synergy was calculated by the formula: (relative stimulation of +/+) ÷ [(relative stimulation of +/–) + (relative stimulation of –/+)].

Figure 2

Cap–poly(A) synergy as a function of mRNA concentration and extract depletion. (A) Ten aliquots of ribosome-depleted RRL prepared under various centrifugation conditions were programmed with the four different versions of pB2-derived mRNAs as described in the legend to Figure 1. Translation products were analysed as described in the legend to Figure 1 and cap–poly(A) synergy in each extract was calculated. Cap–poly(A) synergy is plotted against the translation efficiency of +/– mRNA translation in the particular batch of depleted RRL relative to that observed in standard RRL (residual translation activity; a value of 1.0 reflects retention of 100% translation activity). Differences in preparation of batches of depleted RRL were as follows: centrifugation at 90 000 r.p.m. for 15, 20, 25, 40 or 45 min (aliquots 9, 6, 3, 2 and 1, respectively, counting from the left) or 85 000 r.p.m. for 15 or 35 min (aliquots 10 and 7). Aliquot 8 was prepared by supplementing aliquot 1 with 0.075× ribosome pellet (with respect to starting extract) recovered after centrifugation. Aliquots 4 and 5 were derived by mixing aliquots 3 and 9 in the ratios 95:5 and 90:10, respectively. (B) Ribosome-depleted extract number 4 [marked with an asterisk in (A)] was programmed with the indicated final RNA concentrations of the four different versions of B2 mRNA under the conditions described in the legend to Figure 1. Translation products were analysed and quantified as described in the legend to Figure 1 and cap–poly(A) synergy in each extract was calculated. RNA concentration is plotted against translation efficiency (open triangles, –/– mRNAs; diamonds, +/–; filled triangles, –/+; circles, +/+; left-hand y-axis) and calculated cap–poly(A) synergy (thick line; right-hand y-axis).

Figure 3

Time course of protein synthesis in ribosome-depleted RRL. Ribosome-depleted RRL reactions were programmed with +/–, –/+ or +/+ mRNAs derived from the p0p24 plasmids (final RNA concentration 4 µg/ml) as described in the legend to Figure 1. Aliquots were removed at 15 min intervals and the reactions stopped prior to analysis of translation products as described in the legend to Figure 1. Control reactions programmed with water gave no detectable protein synthesis (data not shown). The yield of translation products for each mRNA (triangles, +/+; diamonds, +/–; circles, –/+) is plotted against time of incubation.

Figure 4

Influence of KCl and MgCl₂ concentrations on cap–poly(A) synergy in ribosome-depleted RRL. Ribosome-depleted RRL was programmed with mRNAs derived from pB2 (final RNA concentration 6.1 µg/ml) transcribed in the form indicated alongside each panel. Translation reactions contained 0.65 mM added MgCl₂ and varying concentrations of added KCl (80–133 mM; left) or 125 mM added KCl and varying concentrations of added MgCl₂ (0.3–1.3 mM; right). Translation products were analysed as described in the legend to Figure 1. The translation efficiencies of the various RNAs (squares, –/–; diamonds, +/–; circles, –/+; triangles, +/+) plotted as a function of salt concentration and the cap–poly(A) synergy calculated at each salt concentration are shown below the two series of panels.

Figure 5

(A) Western blot analysis of equal volumes of RRL or ribosome-depleted RRL was performed as described in Materials and Methods with antibodies against the indicated translation factors. Volumes of 5, 2.5 and 1.25 µl of RRL and ribosome-depleted RRL were analysed to ensure linearity of the immunological responses. For membranes developed with antibodies against eIF4E, eIF4A and PABP, only the lanes loaded with 1.25 µl of each extract are shown. Developed membranes were analysed by densitometry and the intensity of the immunological signal is plotted (in arbitrary units) below each panel. The percentage of each protein remaining in depleted RRL is indicated below each plot (100% is the signal observed with each antibody against the same volume of standard RRL). (B) Ribosome-depleted RRL was programmed with the indicated forms of pB2-derived mRNAs (final RNA concentration 6.1 µg/ml) and supplemented with 20% (v/v) H100 buffer, HeLa cell S10 extract, S100 extract or HS100 extract as indicated (see Materials and Methods; final salt concentrations 125 and 0.8 mM added KCl and MgCl₂, respectively). Translation products were analysed as described in the legend to Figure 1. The translation efficiency, relative stimulation (as compared to the –/– mRNA in each set of conditions) and calculated cap–poly(A) synergy in each extract are indicated.

Figure 6

Sensitivity of polyadenylated and non-polyadenylated capped mRNA translation to cap analogue inhibition in depleted RRL. (A) Schematic representation of the action of rotavirus NSP3 protein, which abolishes the possibility of mRNA circularisation via the cap–eIF4E–eIF4G–PABP–poly(A) tail interaction. (B) RRL translation reactions, supplemented with NSP3 protein in H100 buffer (NSP3 concentration 10 µg/ml in the reaction) or H100 buffer were immunoprecipitated with antibody against eIF4G or preimmune sera (as indicated, + and –). The immunoprecipitates were analysed for the presence of PABP by western blotting. A photograph of the developed membrane is shown. The percentage of PABP in the immunoprecipitate is given below each lane. (C) Ribosome-depleted RRL was programmed with the indicated forms of p0p24-derived mRNAs (final RNA concentration 3.15 µg/ml) and supplemented with H100 buffer or NSP3 protein in H100 buffer (as indicated; final salt concentrations 125 and 0.8 mM added KCl and MgCl₂, respectively). Reactions then received increasing concentrations of cap analogue in H100 buffer (final concentrations from left to right: 0, 0.22, 1.1, 5.5, 11, 22 and 66 µM). Translation products were analysed as described in the legend to Figure 1. The translation efficiencies of the +/+ (diamonds) and +/– (circles) mRNAs, relative to the efficiencies observed in the absence of cap analogue, are plotted against cap analogue concentration. Open and filled symbols represent, respectively, the translation efficiencies in the absence and presence of NSP3.