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A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation

1998; Springer Nature; Volume: 17; Issue: 24 Linguagem: Inglês

10.1093/emboj/17.24.7480

1460-2075

Hiroaki Imataka,

Polyamine Metabolism and Applications

Article15 December 1998free access A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation Hiroaki Imataka Hiroaki Imataka Department of Biochemistry and McGill Cancer Centre, McGill University, Drummond Street 3655, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Alessandra Gradi Alessandra Gradi Department of Biochemistry and McGill Cancer Centre, McGill University, Drummond Street 3655, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Nahum Sonenberg Corresponding Author Nahum Sonenberg Department of Biochemistry and McGill Cancer Centre, McGill University, Drummond Street 3655, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Hiroaki Imataka Hiroaki Imataka Department of Biochemistry and McGill Cancer Centre, McGill University, Drummond Street 3655, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Alessandra Gradi Alessandra Gradi Department of Biochemistry and McGill Cancer Centre, McGill University, Drummond Street 3655, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Nahum Sonenberg Corresponding Author Nahum Sonenberg Department of Biochemistry and McGill Cancer Centre, McGill University, Drummond Street 3655, Montreal, Quebec, Canada, H3G 1Y6 Search for more papers by this author Author Information Hiroaki Imataka1, Alessandra Gradi1 and Nahum Sonenberg 1 1Department of Biochemistry and McGill Cancer Centre, McGill University, Drummond Street 3655, Montreal, Quebec, Canada, H3G 1Y6 *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:7480-7489https://doi.org/10.1093/emboj/17.24.7480 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Most eukaryotic mRNAs possess a 5′ cap and a 3′ poly(A) tail, both of which are required for efficient translation. In yeast and plants, binding of eIF4G to poly(A)-binding protein (PABP) was implicated in poly(A)-dependent translation. In mammals, however, there has been no evidence that eIF4G binds PABP. Using 5′ rapid amplification of cDNA, we have extended the known human eIF4GI open reading frame from the N-terminus by 156 amino acids. Co-immunoprecipitation experiments showed that the extended eIF4GI binds PABP, while the N-terminally truncated original eIF4GI cannot. Deletion analysis identified a 29 amino acid sequence in the new N-terminal region as the PABP-binding site. The 29 amino acid stretch is almost identical in eIF4GI and eIF4GII, and the full-length eIF4GII also binds PABP. As previously shown for yeast, human eIF4G binds to a fragment composed of RRM1 and RRM2 of PABP. In an in vitro translation system, an N-terminal fragment which includes the PABP-binding site inhibits poly(A)-dependent translation, but has no effect on translation of a deadenylated mRNA. These results indicate that, in addition to a recently identified mammalian PABP-binding protein, PAIP-1, eIF4G binds PABP and probably functions in poly(A)-dependent translation in mammalian cells. Introduction Most eukaryotic mRNAs possess a cap structure at the 5′ end and a poly(A) tail structure at the 3′ end. The cap structure is bound by eukaryotic initiation factor (eIF) 4F, which consists of eIF4E, eIF4A and eIF4G. eIF4E is the cap-binding subunit. eIF4A is an RNA-dependent ATPase and ATP-dependent RNA helicase, which, in combination with eIF4B, is thought to unwind the secondary structure in the 5′-untranslated region of the mRNA to facilitate ribosome binding (for reviews, see Merrick and Hershey, 1996; Sonenberg, 1996). eIF4G serves as a scaffold for eIF4E and eIF4A to coordinate their functions. eIF4G also binds eIF3, which associates with the 40S ribosomal subunit (for reviews, see Pain, 1996; Morley et al., 1997; Sachs et al., 1997). The poly(A) tail is bound by the poly(A)-binding protein (PABP), which is necessary for efficient translation (for reviews, see Jacobson, 1996; Sachs et al., 1997). In yeast, eIF4G was found to associate with PABP (Tarun and Sachs, 1996). The PABP-binding site was mapped to an N-terminal region proximal to the eIF4E-binding site, which mediates the poly(A) tail-dependent translation and circularization of the mRNA (Tarun and Sachs, 1996; Tarun et al., 1997; Wells et al., 1998). In plants, PABP was reported to bind to eIF-iso4G and eIF4B. Their interaction with PABP increased the RNA-binding activity of PABP (Le et al., 1997). In mammals, a PABP-binding protein (PAIP-1) was cloned by far Western screening with PABP as a probe (Craig et al., 1998). PAIP-1 bound eIF4A as well as PABP, and enhanced translation in vivo (Craig et al., 1998). Interaction between eIF4G and PABP, however, has not been documented in mammals. We recently have cloned a cDNA for human eIF4GII (Gradi et al., 1998), a closely related functional homologue of eIF4G (hereafter called eIF4GI) (Yan et al., 1992). The open reading frame (ORF) of eIF4GII was found to be N-terminally longer by 158 amino acids relative to the methionine which aligns with the first methionine of the published eIF4GI sequence (Gradi et al., 1998). We suspected that the original cDNA clone of eIF4GI (Yan et al., 1992) contained an intron, because the similarity of this clone to that of eIF4GII in nucleotide sequence stops abruptly at a putative splice acceptor site (Gradi et al., 1998). Consequently, we performed 5′ rapid amplification of cDNA ends (5′ RACE), and extended the N-terminus of eIF4GI by 109 amino acids to form a segment which exhibits significant homology to the corresponding region of eIF4GII (Gradi et al., 1998). Here we show that the extended N-terminal region of human eIF4GI contains a PABP-binding site, and functions in translation in a poly(A)-dependent manner. Results Full-length eIF4G binds PABP Using 5′ RACE, we extended the N-terminal sequence of human eIF4GI by 109 amino acids (Gradi et al., 1998) as compared with the original eIF4GI clone (Yan et al., 1992). However, the new sequence did not contain an in-frame stop codon upstream of the first ATG. Consequently, to extend the 5′ end of human eIF4GI further, we repeated the 5′ RACE, and obtained several extended cDNAs. The first methionine in the ORF of the longest clone was assigned tentatively as the first amino acid of human eIF4GI (Figure 1). It is not completely certain that the assigned methionine is indeed the authentic initiator, since there is still no in-frame stop codon upstream of the first AUG. However, this methionine aligns with the initiator methionine in eIF4GII (Gradi et al., 1998), and is thus likely to be the initiator methionine of eIF4GI. The new ORF of eIF4GI (Figure 1) is 156 amino acids longer than that of Yan et al. (1992). The N-terminally extended sequence was fused to the original cDNA clone (1404 amino acids) (Yan et al., 1992) to construct a cDNA encoding an ORF of 1560 amino acids (accession No. AF104913) with a predicted molecular mass of 171 kDa. The resulting clone is referred to as the extended eIF4GI throughout this manuscript. Figure 1.Alignment of amino acid sequences of the N-terminal region of human eIF4GI and eIF4GII. The pattern-induced multi-sequence alignment program (Smith and Smith, 1992) was used to align amino acid sequences of the N-terminal region of eIF4GI (Yan et al., 1992; Imataka and Sonenberg, 1997; this study) and eIF4GII (Gradi et al., 1998). Identical amino acids are boxed and conservative substitutions are shaded. The first methionine of the original eIF4GI (Yan et al., 1992) is indicated by an arrow. Numbers above amino acids represent positions of truncations used in the text. A schematic representation of the binding sites of eIF4E (Mader et al., 1995), eIF4A and eIF3 (Lamphear et al., 1995; Imataka and Sonenberg, 1997) is shown above the alignment. Download figure Download PowerPoint As the original clone of human eIF4GI (Yan et al., 1992) did not exhibit any binding activity to PABP using several methods, including the yeast two-hybrid system (Craig et al., 1998), co-immunoprecipitation or in vitro binding assays with recombinant proteins (H.Imataka, unpublished data), we wished to determine whether the extended eIF4GI could interact with PABP. The original eIF4GI (Yan et al., 1992) and the extended eIF4GI were tagged with the hemagglutinin (HA) epitope, and expressed in HeLa cells. Cytoplasmic extracts were immunoprecipitated with an anti-HA antibody, and the immunoprecipitated proteins were subjected to Western blotting with anti-PABP, anti-eIF4A or anti-HA antibodies. Much smaller amounts of the HA-extended eIF4GI than those of the original eIF4GI were detected in the immunoprecipitates. [Figure 2A, lower panel; compare lanes 2 and 3; the expression of HA-extended eIF4GI in the whole cell is one-fifth of that of the original eIF4GI, for unknown reasons. In addition, the extended eIF4GI could not be extracted with the buffer used (see Materials and methods) as efficiently as the original eIF4GI, probably because the extended N-terminal region of eIF4GI is rich in hydrophobic amino acids (Figure 1), which causes the protein to sediment with the cell debris. As a consequence, to visualize the extended product, the X-ray film was overexposed.] In spite of this difference, PABP was co-precipitated efficiently with the extended eIF4GI (upper panel, lane 2), while no PABP was precipitated with the original eIF4GI (lane 3) or from control extracts (lane 1). Both versions of eIF4GI bound eIF4A (middle panel, lanes 2 and 3), as the binding sites for eIF4A are contained in both (Imataka and Sonenberg, 1997). Similar results were obtained for eIF4GII. Full-length eIF4GII bound PABP (upper panel, lane 4), while an N-terminally truncated form of eIF4GII, which corresponds to the original eIF4GI in the amino acid alignment, failed to bind PABP (lane 5). eIF4A was associated with both forms of eIF4GII (middle panel, lanes 4 and 5). We expressed the same proteins in 293T cells, and confirmed the specific interaction of the HA-extended eIF4GI and endogenous PABP (data not shown). These results clearly show that the full-length eIF4GI and eIF4GII are able to bind PABP, and the N-terminal region which is missing from the original eIF4GI is essential for PABP binding. Figure 2.Co-immunoprecipitation of eIF4GI and eIF4GII with PABP. (A) HeLa cells infected with vTF7-3 were transfected with vector (pcDNA3-HA) alone (lane 1) or the vector expressing an HA-tagged protein as indicated in each lane (lanes 2–5). Proteins were immunoprecipitated with anti-HA antibody and immunoprecipitates were resolved by SDS–10% PAGE. Western blotting was performed with anti-PABP (upper panel), anti-eIF4A (middle panel) or anti-HA antibody (lower panel). HeLa cell extract (40 μg of protein) was loaded to the left of lane 1. (B) Extracts from uninfected and non-transfected HeLa cells were incubated with pre-immune serum (lane 2) or anti-PABP serum (lane 3). Following precipitation with protein G–Sepharose, bound proteins were resolved by SDS–8% PAGE. Western blotting was performed with anti-eIF4GI (left upper panel), anti-eIF4GII (right upper panel) or anti-PABP antibody (lower panels). One-fiftieth of the extracts used for immunoprecipitation was loaded in lane 1. Download figure Download PowerPoint To determine whether endogenous eIF4GI and eIF4GII are associated with PABP in vivo, cytoplasmic extracts from HeLa cells were used for immunoprecipitation with anti-PABP serum. Both eIF4GI and eIF4GII co-immunoprecipitated with anti-PABP serum (Figure 2B, lane 3), while a pre-immune serum failed to precipitate either PABP or eIF4G (lane 2). About 5% of eIF4GI and eIF4GII in the extract was immunoprecipitated with anti-PABP serum, as determined by laser densitometry. These results indicate that endogenous eIF4GI and eIF4GII are associated with PABP in mammalian cells. We could not perform the reciprocal experiment with an antibody to eIF4G, as the titre of our antibodies was not sufficiently high for immunoprecipitation. PABP-binding site in eIF4GI To localize the PABP-binding site in the extended human eIF4GI, fragments of eIF4GI were N-terminally tagged with glutathione S-transferase (GST) and co-expressed with PABP-HA (C-terminally HA-tagged human PABP) in HeLa cells. A portion of the cell extract was subjected to Western blotting with anti-GST antibody to confirm the expression of the eIF4GI fragments (Figure 3A, B and C, upper panels). The rest of the extract was immunoprecipitated with anti-HA antibody, and immunoprecipitates were used for Western blotting with anti-GST antibody (Figure 3A, B and C, middle panels) or anti-HA antibody (Figure 3A, B and C, lower panels) to determine PABP binding. eIF4GI(1–329) bound PABP to a significant extent (Figure 3A, lane 1), while chloramphenicol acetyltransferase (CAT), which serves as a negative control, did not bind (Figure 3A, lane 6). Progressive N-terminal truncations extending to amino acid 131 led to a graded decrease in the amount of eIF4GI fragments co-precipitated with PABP (Figure 3A, lanes 2–5 and B, lanes 1–3). The N-terminal boundary for PABP binding resides between amino acids 132 and 139, because eIF4GI(132–329) bound PABP, but eIF4GI(139–329) did not (Figure 3B, compare lanes 3 and 4). Similar immunoprecipitation experiments were performed to determine the C-terminal boundary of the PABP-binding site. While eIF4GI(45–160) bound PABP (Figure 3C, lane 3) to the same extent as eIF4GI(45–329) (lane 1), the fragment eIF4GI(45–155) failed to bind PABP (lane 4). Thus, the C-terminal boundary for PABP binding resides between amino acids 155 and 160. The sequence (amino acids 132–160) of eIF4GI is almost identical to the corresponding sequence (amino acids 135–162) of eIF4GII, of which only two amino acids are different, but represent conservative changes, R to K and A to G (see Figure 1). Figure 3.Localization of the PABP-binding site in human eIF4GI. (A) N-terminal deletions. HeLa cells infected with vTF7-3 were co-transfected with pcDNA3-PABP-HA and pcDNA3-GST-eIF4GI fragments indicated in each lane (lanes 1–5) or pcDNA3-GST-CAT (lane 6). One-tenth of the cell extract was used for Western blotting with anti-GST antibody (upper panel). The remaining extract was used for immunoprecipitation with anti-HA antibody. Immunoprecipitates were resolved by SDS–10% PAGE. Western blotting was performed with anti-GST (middle panel) or anti-HA antibody (lower panel). (B) N-terminal boundary. Experiments were as in (A), with plasmids expressing GST fusion proteins indicated in each lane. (C) C-terminal boundary. Experiments were as in (A), with plasmids expressing GST fusion proteins indicated in each lane. (D) eIF4GI(132–160) is the PABP-binding site. HeLa cells infected with vTF7-3 were co-transfected with pcDNA3-FLAG-PABP and pcDNA3-GST-eIF4GI fragments indicated in each lane (lanes 1–3) or pcDNA3-GST (lane 4). One-tenth of the cell extract was used for Western blotting with anti-FLAG antibody (upper panel), and the remaining extract was mixed with glutathione–Sepharose beads. Bound proteins were eluted with reduced glutathione, and were resolved by SDS–12.5% PAGE. Western blotting was performed with anti-FLAG (middle panel) or anti-GST antibody (lower panel). The amino acid sequence of the PABP-binding site in human eIF4GI and the corresponding region of human eIF4GII are aligned at the bottom of the figure. Identical amino acids are boxed and conservative substitutions are shaded. (E) eIF4GI(132–160) binds endogenous PABP. GST (lane 1) or GST–eIF4GI(132–160) (lane 2) was expressed as in (D), but without co-expression of FLAG-PABP. The cell extract was mixed with glutathione–Sepharose beads. Bound proteins eluted with reduced glutathione were subjected to Western blotting with anti-PABP (upper panel) or anti-GST antibody (lower panel). HeLa cell extract (40 μg protein) was loaded to the left of lane 1. Download figure Download PowerPoint To determine whether the 29 amino acid segment (132–160) of eIF4GI alone has the capacity to bind PABP, a GST fusion protein, GST–eIF4GI(132–160), was co-expressed with FLAG-PABP in HeLa cells. A portion of the cell extract was subjected to Western blotting with anti-FLAG antibody to confirm the expression of FLAG-PABP (Figure 3D, upper panel). The rest of the extract was incubated with glutathione–Sepharose beads, and bound proteins were used for Western blotting with anti-FLAG (middle panel) or anti-GST antibody (lower panel). As expected, FLAG-PABP was co-precipitated with GST–eIF4GI(45–160), but not with GST–eIF4GI(45–155) (Figure 3D, compare lanes 1 and 2). While GST alone did not interact with FLAG-PABP (lane 4), the GST–eIF4GI(132–160) amino acid fusion protein bound FLAG-PABP (lane 3). To examine whether the 29 amino acid segment binds to the endogenous PABP, GST or GST–eIF4GI(132–160) was expressed in HeLa cells. The cell extract was incubated with glutathione–Sepharose beads, and the bound proteins were used for Western blotting with anti-PABP (Figure 3E, upper panel) or anti-GST (lower panel). The endogenous PABP was co-precipitated with GST–eIF4GI(132–160), but not with GST (Figure 3E, compare lanes 1 and 2). Based on these results, we conclude that the 29 amino acid segment (132–160) of eIF4GI contains the core PABP-binding site. Although we did not delimit the PABP-binding domain further, it is likely that it constitutes the smallest binding motif, because of the high conservation between amino acids 132–139 of eIF4GI and 135–162 of eIF4GII (Figure 3D). The region (amino acids 45–131) of eIF4GI might increase the binding affinity of the binding domain for PABP by stabilizing the structure. Interestingly, there is no significant homology in the amino acid sequence between the PABP-binding sites of human and yeast eIF4G (Tarun et al., 1997). This will be addressed in the Discussion. eIF4GI-binding site in PABP PABP consists of four highly evolutionarily conserved RNA recognition motifs (RRMs) and a less conserved C-terminal region (Sachs et al., 1986; Burd et al., 1991) (Figure 4A). In the yeast PABP, a region composed of RRM1 and RRM2 is essential for interaction with eIF4G (Kessler and Sachs, 1998). As the sequence of the PABP-binding site in eIF4G is not conserved between yeast and human, we wished to determine the binding site of human eIF4GI in human PABP. Initially, two fragments of PABP, an N-terminal region, PABP(N), which contains the four RRMs, and a C-terminal region, PABP(C), which is devoid of the RRMs, were FLAG tagged, and co-expressed with HA-eIF4GI(1–329) in HeLa cells. Subsequently, fragments encompassing the separate RRMs and various combinations of the RRMs were also generated. The constructs are shown schematically in Figure 4A. A portion of the extract was subjected to Western blotting with anti-FLAG antibody to confirm the expression of the PABP fragments (Figure 4B and C, upper panels). The rest of the extract was immunoprecipitated with anti-HA antibody, and immunoprecipitates were used for Western blotting with anti-FLAG (Figure 4B and C, middle panels) or anti-HA antibody (Figure 4B and C, lower panels) to determine which fragment of PABP bound eIF4GI. The N-terminal fragment composed of RRMs 1, 2, 3 and 4 clearly bound eIF4GI(1–329) (Figure 4B, lane 1), while the C-terminal region of PABP (lane 2) and eIF4E, which served as a negative control (lane 3), failed to bind, indicating that human eIF4GI binds to the N-terminal RRM region of PABP. To delimit the eIF4GI-binding site in the N-terminal region, pairs of RRMs (RRMs 1–2, RRMs 2–3 or RRMs 3–4) or single RRMs (RRM1 or RRM2) were fused to the C-terminal region of PABP (Figure 4A) and tested for binding to eIF4GI. RRMs 1–2–C (Figure 4C, lane 2) bound eIF4GI(1–329) to the same extent as full-length PABP (lane 1), while RRMs 2–3–C bound eIF4GI with a considerably decreased affinity (lane 3). Other PABP fragments and luciferase (negative control) did not bind (lanes 4–7). Finally, to examine whether RRMs 1–2 is able to bind eIF4GI(132–160), GST or GST–eIF4GI(132–160) was co-expressed with FLAG-RRMs 1–2 in HeLa cells. A portion of the cell extract was subjected to Western blotting with anti-FLAG antibody to confirm the expression of FLAG-RRMs 1–2 (Figure 4D, upper panel). The rest of the extract was incubated with glutathione–Sepharose beads, and bound proteins were used for Western blotting with anti-FLAG (middle panel) or anti-GST antibody (lower panel). FLAG-RRMs 1–2 was able to interact with GST–eIF4GI(132–160) (lane 2), but not with GST alone (lane 1). These results demonstrate that, similarly to the yeast PABP–eIF4G interaction (Kessler and Sachs, 1998), a region composed of RRM1 and RRM2 contains the eIF4G-binding site. As RRMs 2–3 showed a relatively weak but significant affinity for eIF4GI, RRM2 might be the most important region for binding eIF4GI, as was shown for yeast (Kessler and Sachs, 1998). However, RRM2 alone exhibited little binding affinity for eIF4GI (Figure 4C, lane 6). Presumably, a combination of the first two RRMs is necessary for an appropriate structural conformation to facilitate eIF4G binding. We failed to detect endogenous eIF4Gs co-immunoprecipitated with PABP(RRMs 1–2–C)-HA or with PABP-HA, probably because endogenous PABP is much more abundant than endogenous eIF4G (see Discussion). Thus, eIF4G is most likely saturated with endogenous PABP, and the expressed PABP could not replace the endogenous PABP efficiently for eIF4G-binding. Figure 4.Localization of the eIF4GI-binding site in PABP. (A) Schematic representation of PABP mutants examined in (B) and (C). (B) eIF4GI binds the N-terminal region (RRMs 1–4) of PABP. HeLa cells infected with vTF7-3 were co-transfected with pcDNA3-HA-eIF4G(1–329) and pcDNA3-FLAG-PABP(RRMs 1–4) (lane 1), -PABP(C) (lane 2) or -eIF4E (lane 3). One-twentieth of the cell extract was used for Western blotting with anti-FLAG antibody (upper panel). The remaining extract was used for immunoprecipitation with anti-HA antibody. Immunoprecipitates were resolved by SDS–10% PAGE. Western blotting was performed with anti-FLAG (middle panel) or anti-HA antibody (lower panel). (C) eIF4GI binds RRMs 1–2. Experiments were as in (A), with plasmids expressing FLAG-tagged proteins indicated in each lane. (D) RRMs 1–2 binds eIF4GI(132–160). GST (lane 1) or GST–eIF4GI(132–160) (lane 2) was co-expressed with FLAG-RRMs 1–2 as in Figure 3D. One-tenth of the cell extract was used for Western blotting with anti-FLAG antibody (upper panel), and the remaining extract was mixed with glutathione–Sepharose beads. Bound proteins eluted with reduced glutathione were subjected to Western blotting with anti-FLAG (middle panel) or anti-GST antibody (lower panel). Download figure Download PowerPoint Functional analysis of the eIF4GI N-terminal region To determine whether the PABP binding to the N-terminal region of eIF4G is functionally significant, a recombinant N-terminal region (amino acids 1–204) of eIF4GI, which contains the PABP-binding site, was prepared as a GST fusion protein, GST–eIF4GI(1–204). As a control, we also prepared GST–eIF4GI(1–204:mut), in which amino acids 134–138, KRERK, in the PABP-binding site were converted to alanines. To determine the binding of these eIF4GI fragments to PABP, they were mixed with PABP(RRMs 1–4)-His protein, and precipitated with glutathione–Sepharose beads. Bound proteins were subjected to Western blotting with anti-GST (Figure 5A, upper panel) or anti-His antibody (lower panel). While PABP(RRMs 1–4)-His failed to associate with GST (Figure 5A, lower panel, lane 1) or GST–eIF4GI(1–204:mut) (lane 3), GST–eIF4GI(1–204) interacted with PABP(RRMs 1–4)-His to a significant extent (lane 2). This binding assay was performed without addition of poly(A) RNA. In similar binding experiments using yeast eIF4G and PABP, the presence of poly(A) RNA was essential for the interaction between these proteins (Tarun and Sachs, 1996). Figure 5.Functional analysis of the N-terminal region of eIF4GI. (A) In vitro binding of the N-terminal sequence of eIF4GI to PABP. PABP(RRMs 1–4)-His was incubated with GST (lane 1), GST–eIF4GI(1–204) (lane 2) or GST–eIF4GI(1–204:mut) (lane 3) immobilized on glutathione–Sepharose beads. Bound proteins were eluted with reduced glutathione, and resolved by SDS–12.5% PAGE. Western blotting was performed with anti-GST (upper panel) or anti-His (lower panel) antibody. One-fifth of the input PABP(RRMs 1–4)-His was loaded to the left of lane 1. (B) Effects of the N-terminal region on poly(A)-dependent translation. Buffer (lanes 1 and 5) (1 μl), GST (lanes 2 and 6), GST–eIF4G(1–204) (lanes 3, 8, 9 and 10) or GST–eIF4G(1–204:mut) (lanes 4 and 7) (2, 4 or 6 μg) (1 μl) was added to a rabbit reticulocyte lysate (10 μl). After incubation on ice 30 min, the lysate was programmed with capLUC (lanes 1–4) or capLUCpA RNA (lanes 5–10) (1 μl, 60 ng). The translation reaction mixture was incubated at 30°C for 30 min. Luciferase activity was measured using a luminometer. The luciferase activity of the lysate programmed with capLUC in the presence of buffer alone (lane 1) was set at 100%. Error bars denote the standard error of four independent experiments. Download figure Download PowerPoint Next, we wished to examine whether the N-terminal region of eIF4GI containing the PABP-binding site is able to act as an inhibitor of poly(A)-dependent translation. A rabbit reticulocyte lysate was mixed with GST, GST–eIF4G(1–204), GST–eIF4GI(1–204:mut) or buffer alone, and programmed with capped luciferase RNA (capLUC) or capped and poly(A)-tailed luciferase RNA (capLUCpA) for in vitro translation followed by monitoring of luciferase activity. None of the recombinant proteins exhibited any effects on translation of capLUC (Figure 5B, lanes 1–4). As observed by others (Grossi de Sa et al., 1988; Munroe and Jacobson, 1990), the presence of a poly(A) tail increased translation of the mRNA in the rabbit reticulocyte lysate by ∼2-fold (the average of four experiments with a standard error of 10%; Figure 5B, compare lanes 1 and 5). The functional half-life (Gallie, 1991; Tarun and Sachs, 1995) for capLUC and capLUCpA was 14 ± 2 and 15 ± 2 min (the mean ± the standard error of three independent experiments), respectively, indicating that the stimulation by the poly(A) tail was not attributable to a difference in mRNA stability. When luciferase RNAs (capLUC and capLUCpA) and CAT RNAs (capCAT and capCATpA) were translated in the presence of [35S]methionine, the poly(A) tail increased incorporation of the radioactivity into the translated products by ∼2-fold for both luciferase and CAT (data not shown), indicating that the observed effect is independent of a reporter mRNA. Moreover, translation of capLUCpA was inhibited more strongly (77%) by incubation with poly(A) (10 ng/μl) than with poly(C) (40% inhibition at 10 ng/μl), while translation of capLUC was inhibited with poly(A) and poly(C) to the same extent (50%, at 10 ng/μl) (data not shown). These observations validate the use of the rabbit reticulocyte lysate and luciferase mRNA for the functional analysis. The translational enhancement by the poly(A) tail was decreased proportionally by increasing amounts of GST–eIF4G(1–204) (2–6 μg) (lanes 8, 9 and 10). Addition of GST alone showed no effect on the poly(A)-dependent translation (lane 6). The effect of GST–eIF4G(1–204) on translation of the poly(A) RNA is explained by the disruption of the interaction between eIF4G and PABP, because GST–eIF4G(1–204:mut), which failed to bind PABP, decreased translation of capLUCpA only slightly (10%) (lane 7). We quantified the amount of PABP in the reticulocyte lysate to be 0.2 μg/10 μl. Considering the molecular masses of PABP (70 kDa) and GST–eIF4GI(1–204) (47 kDa), 6 μg of GST–eIF4GI(1–204), which was required to abrogate the effect of the poly(A) tail (compare lanes 8–10), corresponds to a 45-fold molar excess over PABP. Thus, to suppress poly(A) tail-dependent translation, a large excess of GST–eIF4GI(1–204) over endogenous PABP is required. The GST portion of GST–eIF4GI(1–204) might hinder this fusion protein from gaining access to PABP which is associated with the full-length eIF4Gs. We attempted to neutralize the effect of GST–eIF4GI(1–204) (6 μg) by adding recombinant PABP (0.2–1.4 μg) to the lysate, but failed to restore translation, because the excess GST–eIF4GI(1–204) in the system should readily neutralize exogenously added PABP. These functional assays suggest that the PABP–eIF4G interaction is required for the poly(A)-dependent translation, although an experiment using full-length recombinant eIF4G, which could not be obtained, is required to prove this. Taken together, our results show that the N-terminal region of human eIF4GI binds PABP, and probably functions to mediate the translational enhancement by the poly(A) tail. Discussion We have shown that human eIF4G interacts with PABP in a functionally significant manner. PAIP-1, a recently identified mammalian PABP-binding protein, binds eIF4A, and stimulates translation (Craig et al., 1998). Thus, mammalian cells possess dual systems, PABP–PAIP-1 and PABP–eIF4G, to effect poly(A)-dependent translation. Irrespective of whether the binding sites of eIF4G and PAIP-1 in PABP are overlapping, both systems could operate in a non-competitive manner in vivo, since PABP is as abundant as eIF4A (Görlach et al., 1994), while eIF4G is six times less abundant than eIF4A (Duncan et al., 1987), and PAIP-1 appears to be present at 6-fold lower amounts