The role of ABCE1 in eukaryotic posttermination ribosomal recycling

Abstract

After termination, eukaryotic 80S ribosomes remain associated with mRNA, P-site deacylated tRNA, and release factor eRF1 and must be recycled by dissociating these ligands and separating ribosomes into subunits. Although recycling of eukaryotic posttermination complexes (post-TCs) can be mediated by initiation factors eIF3, eIF1, and eIF1A (Pisarev et al., 2007), this energy-free mechanism can function only in a narrow range of low Mg(2+) concentrations. Here, we report that ABCE1, a conserved and essential member of the ATP-binding cassette (ABC) family of proteins, promotes eukaryotic ribosomal recycling over a wide range of Mg(2+) concentrations. ABCE1 dissociates post-TCs into free 60S subunits and mRNA- and tRNA-bound 40S subunits. It can hydrolyze ATP, GTP, UTP, and CTP. NTP hydrolysis by ABCE1 is stimulated by post-TCs and is required for its recycling activity. Importantly, ABCE1 dissociates only post-TCs obtained with eRF1/eRF3 (or eRF1 alone), but not post-TCs obtained with puromycin in eRF1's absence.

Figure 1. Association of Post-TCs with eRF1 and their recycling by eIFs at Different Mg²⁺ Concentrations

(A) Structure of MVHL-STOP mRNA. (B) Dissociation of pre-TCs, assembled on MVHL-STOP mRNA with [³²P]60S subunits and native Leu-tRNA^Leu after incubation with eRFs, eIFs, and puromycin at indicated Mg²⁺ concentrations, assayed by SDG centrifugation. (C) Toeprinting analysis of ribosomal complexes obtained by incubating pre-TCs assembled on MVHL-STOP mRNA using native tRNA^Leu with eRFs at 2.5 mM Mg²⁺. Lanes C, T, A, and G depict cDNA sequences corresponding to MVHL-STOP mRNA. (D) Association of [³²P]eRF1 with post-TCs assembled on MVHL-STOP mRNA with native Leu-tRNA^Leu at indicated Mg²⁺ concentrations, assayed by SDG centrifugation. (E) Dissociation of pre-TCs, assembled on MVHL-STOP mRNA with [³²P]60S subunits and transcript Leu-tRNA^Leu after incubation with eRFs, eIFs, and puromycin at 1 mM Mg²⁺, assayed by SDG centrifugation. (F) Association of [³²P]eRF1 with post-TCs assembled on MVHL-STOP mRNA with native or transcript Leu-tRNA^Leu at 1 mM Mg²⁺, assayed by SDG centrifugation. (G) Toeprinting analysis of ribosomal complexes obtained by incubating pre-TCs assembled on MVHL-STOP mRNA using transcript tRNA^Leu with eRF1 at 1 mM Mg²⁺. Lanes C, T, A, and G depict cDNA sequences corresponding to MVHL-STOP mRNA. Upper fractions were omitted for clarity from (B) and (D)–(F).

Figure 2. Dissociation of post-TCs by ABCE1

(A) Left panel - scheme for purification of ABCE1 from RRL, right panel - purified ABCE1 resolved by SDS-PAGE. (B-F) Dissociation of pre-TCs, assembled on MVHL-STOP mRNA with [³²P]60S subunits and (B-E) native or (F) transcript Leu-tRNA^Leu, after incubation with different combinations of eRFs, ABCE1, eIFs and unlabeled 60S subunits at indicated Mg²⁺ concentrations, assayed by SDG centrifugation. Upper fractions were omitted for clarity.

Figure 3. Dissociation of tRNA and mRNA from recycled 40S subunits

(A-D) Ribosomal association of (A) [³²P]tRNA^Leu and (B-D) [³²P]MVHL-STOP mRNA after incubating pre-TCs with eRFs, ABCE1 and eIFs at 2.5 mM Mg²⁺, as indicated, assayed by SDG centrifugation. (E, F) Toe-print analysis of ribosomal complexes, obtained by incubating pre-TCs, assembled on MVHL-STOP mRNA using native Leu-tRNA^Leu, with combinations of eRFs, ABCE1 and eIFs at indicated Mg²⁺ concentrations. Lanes C, T, A, G depict cDNA sequences corresponding to MVHL-STOP mRNA. The positions of full-length cDNA and of toe-prints corresponding to ribosomal complexes are indicated. (G) Influence of ABCE1 on association of [³²P]eRF1 with post-TCs at different Mg²⁺ concentrations, assayed by SDG centrifugation. Upper fractions were omitted for clarity from panels A, G.

Figure 4. Ribosomal recycling is impaired in ABCE1-silenced HeLa cell extract: Dissociation of post-TCs by ABCE1 requires eRF1

(A) Expression of ABCE1 in HeLa cells transfected with control (GFP) or ABCE1 siRNAs, assayed by western blotting. (B) Influence of ABCE1 silencing on protein synthesis assayed by the translation efficiency of a capped, polyadenylated luciferase reporter mRNA in control (GFP) and ABCE1-silenced HeLa cell extracts. (C) Toe-print analysis of recycling of post-TCs, assembled on the MVHL-STOP mRNA using the in vitro reconstituted translation system, in control or ABCE1-silenced HeLa cell extracts. Lanes C, T, A, G depict cDNA sequences corresponding to MVHL-STOP mRNA. The positions of full-length cDNA and of toe-prints corresponding to post-TCs are indicated. (D) Dissociation by ABCE1 of post-TCs, obtained by incubation of pre-TCs assembled on MVHL-STOP mRNA using [³²P]60S subunits with eRF1/eRF3, eRF1 alone, or puromycin, assayed by SDG centrifugation. (E) Toe-print analysis of ribosomal complexes, obtained by incubating pre-TCs, assembled on MVHL-STOP mRNA using native Leu-tRNA^Leu, with combinations of eRFs, puromycin, ABCE1 and eIFs at 2.5 mM Mg²⁺. Lanes C, T, A, G depict cDNA sequences corresponding to MVHL-STOP mRNA. The positions of full-length cDNA and of toe-prints corresponding to ribosomal complexes are indicated. (F) Interaction between ABCE1 and eRF1 assayed by co-immunoprecipitation. Anti-ABCE1 antibodies with/without ABCE1 were bound to protein A sepharose, which was then incubated with eRF1. Proteins bound to the matrix were further analyzed by western blotting using anti-eRF1 (upper panel) and anti-ABCE1 (lower panel) antibodies.

Figure 5. Nucleotide-dependence of ribosomal association of ABCE1

(A-C) Association of ABCE1 with (A) 40S subunits, 43S complexes and 80S ribosomes, (B) 60S subunits and (C) pre-TCs assembled with native or transcript Leu-tRNA^Leu and incubated with combinations of wt eRF1, eRF1(AGQ), eRF3 and puromycin, in the presence/absence of nucleotides, as indicated. Ribosomal peak fractions obtained by SDG centrifugation were analyzed by western blotting using anti-ABCE1 antibodies. (D) Association of ABCE1 and eRF1 with individual 80S ribosomes in the absence of nucleotides assayed by SDG centrifugation and western blotting of peak ribosomal fractions using anti-ABCE1 (left panel) and anti-eRF1 (right panel) antibodies. (E) Association of [³²P]eRF1 with 80S ribosomes in the presence/absence of ABCE1 and nucleotides as indicated, assayed by SDG centrifugation. Upper fractions were omitted for clarity. (F) Toe-print analysis of ribosomal complexes obtained by incubating pre-TCs, assembled on MVHL-STOP mRNA using native tRNA^Leu, with eRF1/eRF3 and SDG-purified 80S/eRF1/ABCE1 complexes (panel E). Toe-prints corresponding to ribosomal complexes are indicated.

Figure 6. NTPase activity of ABCE1 is required for its function in ribosomal recycling

(A) Thin-layer chromatography analysis of ABCE1's NTPase activity in the presence/absence of 80S ribosomes. The positions of [α³²P]-NDPs are indicated. (B) Time courses of GTP hydrolysis by ABCE1 in the presence/absence of pre-TC, pre-TC/eRF1(AGQ), pre-TC/eRF1(wt), or pre-TC/eRF1(wt)/eIF6, as indicated. 15 μl reaction mixtures containing 0.5 pmol ABCE1, 0.33 μM [γ-³²P]GTP and combinations of 0.5 pmol pre-TC, 10 pmol eRF1(AGQ), 10 pmol eRF1(wt) and 10 pmol eIF6, were incubated at 37°C. Aliquots were removed after 2-30 minutes. GTP hydrolysis in the presence of both ABCE1 and pre-TCs (upper panels) was corrected to take into account the intrinsic GTPase activity of pre-TCs (lower panels). (C-E) Dissociation by ABCE1 of post-TCs, obtained by incubation of pre-TCs assembled on MVHL-STOP mRNA using native Leu-tRNA^Leu and [³²P]60S subunits with (C, D) eRF1, or (E) eRF1/eRF3, depending on the presence/absence of nucleotides as indicated, assayed by SDG centrifugation. (F) Toe-print analysis of ribosomal complexes obtained by incubating pre-TCs assembled on MVHL-STOP mRNA using transcript tRNA^Leu with eRF1, ABCE1, eIFs and nucleotides, as indicated. The positions of full-length cDNA and of toe-prints corresponding to ribosomal complexes are indicated. (G) Association of [³²P]eRF1 with post-TCs, assembled on MVHL-STOP mRNA with transcript Leu-tRNA^Leu, depending on the presence/absence of ABCE1 and nucleotides, assayed by SDG centrifugation. Upper fractions were omitted for clarity in panels C-E, G.

Figure 7. Model of ribosomal recycling by ABCE1

ABCE1 binds to post-TCs containing eRF1 (or eRF1/eRF3, if eRF3 remains associated with ribosomal complexes), and after hydrolyzing ATP promotes their dissociation into 60S subunits and tRNA- and mRNA-bound 40S subunits.