Cloning and Characterization of Human eIF4E Genes*

Mingxing Gao, Wojciech RychlikDagger , and Robert E. Rhoads§

From the Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results & Discussion
References

Two human eukaryotic initiation factor 4E (eIF4E) genes were isolated and characterized from placental and chromosome 4-specific genomic libraries. One of the genes (EIF4E1) contained six introns, but the other gene (EIF4E2) was intronless, flanked by Alu sequences and 14-base pair (bp) direct repeats, and terminated by a short poly(A) stretch, all characteristics of retrotransposons. Numerous additional intronless eIF4E pseudogenes were found, but unlike EIF4E2, all contained premature in-frame stop codons. The entire EIF4E1 gene spanned >50 kilobase pairs. The coding regions of these two genes differed in four nucleotide residues, resulting in two amino acid differences in the predicted proteins. The promoter of EIF4E1 has been characterized previously. The putative promoter of EIF4E2 contained no TATA box but did contain a transcription initiator region (Inr) and numerous other sequence motifs characteristic of regulated promoters. EIF4E2 contained only two of the three polyadenylation signals present in EIF4E1. Evidence for transcription of both genes was obtained from primer extension, S1 mapping, ribonuclease protection, and reverse transcriptase-polymerase chain reaction experiments. Transcription was found to initiate 19 bp upstream of the translational initiation codon in the case of EIF4E1 and 80 bp in the case of EIF4E2. The two genes were differentially expressed in four human cell lines, Wish, Chang, K562, and HeLa.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

The best understood mechanisms for the regulation of protein synthesis involve modifications in the levels or activities of the initiation factors (1-2). Changes involving eukaryotic initiation factor (eIF)1 2 and associated proteins affect binding of the initiator tRNA to the 40 S ribosomal subunit and occur in response to heat shock, virus infection, deprivation of nutrients, and other conditions. Changes in the eIF4 factors affect binding of mRNA to the 43 S initiation complex and occur in response to mitogens, fertilization, and other conditions. The eIF4 factors consist of the ATP-dependent RNA helicase eIF4A (3, 4), the RNA-binding protein eIF4B (5, 6), the cap-binding protein eIF4E (7), and eIF4G, which has specific binding sites for eIF3, eIF4A, eIF4E, and the poly(A)-binding protein (8-10).

Mammalian eIF4E is a 25-kDa protein of known three-dimensional structure (11) which binds to the mRNA cap (7), to eIF4G (3, 4), to eIF4A (12), and to the eIF4E-binding proteins (13). Its ability to function in protein synthesis is regulated by at least three processes. First, the phosphorylation of eIF4E correlates positively with the rate of translation in a large number of systems (1) and increases the protein's affinity for cap analogues by 3- to 4-fold (14). Second, eIF4E availability is regulated by eIF4E-binding proteins, the phosphorylation of which, in response to insulin and other mitogens, releases them from eIF4E and permits eIF4E binding to eIF4G (13). Third, eIF4E levels are regulated at the transcriptional level. eIF4E mRNA is increased by overexpression of c-Myc as well as transformation of cells by v-Src and v-Abl (15). eIF4E mRNA levels are also elevated in a variety of cells that have been oncogenically transformed by in vivo transfection, viral infection, or chemical mutagenesis (16).

Changes in the intracellular levels of eIF4E have a profound effect on cellular growth control. Ectopic overexpression of eIF4E leads to accelerated cell growth (17), transformation in culture and tumorigenesis in nude mice (18), prevention of apoptosis in growth factor-restricted fibroblasts (19), and elevated intracellular levels of growth-regulated proteins such as cyclin D1, c-Myc, ornithine decarboxylase, ornithine aminotransferase, P23, vascular endothelial growth factor, and fibroblast growth factor (20-26). Reduction in intracellular eIF4E levels by expression of antisense RNA (27) results in phenotypic reversal of ras-transformed fibroblasts (28-29). Naturally occurring breast (30-31) and head-and-neck (32) tumors express elevated levels of eIF4E. The eIF4E gene is increasingly being referred to as a proto-oncogene (30-34).

Previous studies have resulted in the cloning and sequencing of human eIF4E cDNA (35) and a 1.4-kb fragment from the 5'-end of the eIF4E gene (36). To shed more light on the expression of eIF4E, especially at the transcriptional level, we have determined the entire gene structure for human eIF4E. Surprisingly two genes were found, one containing introns and the other not. Furthermore, both genes appear to be expressed in human cells, at least at the transcriptional level.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Materials-- Two human placental genomic libraries in bacteriophage lambda  vectors (EMBL3-SP6/T7 and lambda FIX II) were purchased from CLONTECH (Palo Alto, CA) and Stratagene (La Jolla, CA), respectively. Three human chromosome 4-specific genomic libraries in Charon 21 (LLO4NSO2 and LAO4NSO1) and Charon 40 (LAO4NLO1) and one chromosome 20-specific genomic library in Charon 21 (LL20NSO1) were purchased from the ATCC (Rockville, MD). Bacterial strains NM538 and LE392 were purchased from Stratagene (La Jolla, CA). Restriction endonucleases and the DNA Cycle Sequencing System were purchased from Promega (Madison, WI). Radioisotopes (>5000 Ci/mmol) were purchased from ICN (Costa Mesa, CA). The Geneclean kit was purchased from BIO 101, Inc. (Vista, CA).

Human Cell Lines-- Wish cells, derived from amnion tissue, Chang cells from conjunctiva, K562 cells from chronic myelogenous leukemia, and HeLa cells from epithelioid carcinoma of the cervix were purchased from the ATCC. All cell lines were grown in Dulbecco's modified Eagle's medium with 10% calf serum in 5% CO2 at 37 °C and harvested after 2-3 days.

Screening of Human Genomic DNA Libraries-- Recombinant phage were propagated in the host bacterial strains NM538 and LE392 using standard protocols (37). Plaques were screened by colony hybridization and/or PCR using primers that spanned intron/exon junctions. For screening by colony hybridization, a plasmid (pTCEEC) containing human eIF4E cDNA (38) and PCR fragments derived from it were labeled with [alpha -32P]dCTP by nick translation. Prehybridization was performed in 50% formamide at 42 °C for 2 h, radiolabeled probes were added, and hybridization was carried out for >= 16 h. Membranes were washed twice in 2 × SSC, 0.2% SDS at room temperature for 30 min each and once in 0.1 × SSC, 0.2% SDS at 65 °C for 30 min, where 1 × SSC is 0.15 M sodium chloride and 15 mM sodium citrate. The filters were exposed to Kodak x-ray film or analyzed with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For screening by PCR, the library phage lysates were divided into 50 pools (106 plaque-forming units/each), and 1 µl of phage lysate from each pool was used as a PCR template. The positive pools were plated and allowed to grow 8 h, and the agar was again subdivided into 50 pools and screened by PCR. This process was performed a total of three to four times until a single positive plaque was obtained. DNA from selected recombinant phage was prepared from bacterial lysates (37). DNA inserts were mapped with restriction enzymes, and those containing exons were identified by Southern blotting. Exon-containing restriction fragments were excised from agarose gels, subcloned into pBluescript II (-) (Stratagene), and sequenced to identify exon/intron junctions.

Screening of the EMBL3-SP6/T7 library using radiolabeled human eIF4E cDNA yielded positive plaques corresponding to an intronless form of the eIF4E gene, here named EIF4E2. Screening of the same library with a hybridization probe synthesized with primers 9 and 10 (Table I) yielded the recombinant phage lambda 4E1-A. Two human chromosome-specific genomic libraries in Charon 21 were screened using radiolabeled human eIF4E cDNA, one from chromosome 4 and one from chromosome 20. No positive plaques were obtained from the chromosome 20-specific library, but a number of positive plaques were obtained from the chromosome 4-specific genomic library and were termed lambda 4E1-B, lambda 4E1-C, and lambda 4E1-D. A second chromosome 4-specific Charon 21 library was screened with cDNA probes and yielded lambda 4E1-E. The LAO4NLO1 library was screened by PCR as described above using primers 3 and 7 and yielded lambda 4E1-F. The same strategy was applied to this library but with primers 11 and 12, leading to the isolation of lambda 4E1-G, and to the lambda FIX II library with primers 1 and 8 leading to lambda 4E1-H.

                              
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Table I
Oligodeoxynucleotide primers used for PCR, RT-PCR, primer extension, ribonuclease protection, and S1 nuclease mapping

Southern Blot Analysis-- DNA from purified positive plaques (1 µg) was separated on agarose gels and transferred to nitrocellulose (37). DNA probes were labeled with 32P by nick translation. Prehybridization and hybridization were performed as described above for colony hybridization.

DNA Sequence Analysis-- DNA fragments were subcloned into pBluescript II vectors, and sequences were determined by dideoxy chain termination (39) using SK and KS primers (Stratagene) as well as exon-specific oligonucleotide primers. For both EIF4E1 and EIF4E2, the numbering system is based on human eIF4E cDNA (35), i.e. the location of the first ATG in the coding region is designated +1, with upstream nucleotides having negative numbers and downstream nucleotides positive numbers. Nucleotides in introns are not numbered.

RNA Isolation-- Total RNA was prepared from either human placental tissue or human cell lines by the guanidine thiocyanate method (40). In the latter case, ~107 cells were washed with phosphate-buffered saline before homogenization.

Primer Extension Analysis-- Oligonucleotide primers complementary to eIF4E mRNA were end-labeled with T4 polynucleotide kinase (Promega) and [gamma -32P]ATP (37). Primer (300,000 cpm) and total RNA (~100 µg) were heated at 90 °C for 5 min in 30 µl of Hybridization Buffer (80% formamide, 40 mM PIPES, pH 6.4, 400 mM NaCl, and 1 mM EDTA) and then incubated at 30 °C for 16 h. Hybrids were precipitated in ethanol and dissolved in 30 µl of 50 mM Tris-HCl, pH 8.3, 7.5 mM MgCl2, 0.5 mM dNTP, 2 mM dithiothreitol, and 800 units/ml RNasin. Extension was produced by 400 units/ml Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) for 1 h at 42 °C. After ethanol precipitation, products were separated on sequencing gels.

S1 Analysis-- A single-stranded antisense DNA probe corresponding to the sequence between -1000 and +25 of EIF4E1 was produced by asymmetric PCR (41) from the recombinant phage lambda 4E1-H using primers 1 and 4 (Table I). The DNA was electrophoretically separated on 1.2% agarose gels, purified using a Geneclean kit, and then end-labeled with T4 polynucleotide kinase (Promega) and [gamma -32P]ATP (37). Total RNA (~100 µg) was hybridized with the probe (50,000-100,000 cpm) at 30 °C for 16 h in 30 µl of Hybridization Buffer and then digested with 150-200 units of S1 nuclease (Sigma) for 1 h at 30 °C. Protected DNA fragments were separated on 8% sequencing gels (37).

Ribonuclease Protection Analysis-- The sequence between -312 and +184 of EIF4E2 was amplified from phage lambda 4E2 by PCR using primers 2 and 3 (Table I). PCR was performed using the following program: after an initial heating step (95 °C, 10 min), 2 units of Taq DNA polymerase were added to the 100-µl reaction, after which 35 cycles of PCR were carried out (95 °C, 30 s; 57 °C, 30 s; 72 °C, 2 min). The amplified PCR product was purified by agarose gel electrophoresis followed by treatment with Geneclean and then subcloned into the pGEM-T vector (Promega). RNA was transcribed in vitro from pGEM-T linearized by EcoRI and radiolabeled with [32P]UTP with a riboprobe transcription system (Promega). A control RNA was transcribed in vitro from HindIII-linearized pTCEEC. Ribonuclease protection assays were performed as described previously (40).

RT-PCR-- Total RNA from human placental tissue (100 µg) was treated with RNase-free DNase (Promega) at 37 °C for 30 min. After inactivation of the DNase by heating at 70 °C for 15 min, the RNA was hybridized with primer 3 (Table I) in Hybridization Buffer at 30 °C overnight. Primer extension was performed as described above. RNA was removed by DNase-free RNase (Promega), and the remaining DNA from 1 µl of the primer extension reaction mixture was amplified by PCR as described above, except that the annealing temperature was 65 °C.

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

Screening of human genomic libraries yielded two genes capable of encoding eIF4E, one of which (EIF4E1) contained introns and one of which (EIF4E2) did not (Fig. 1). Although a previous study reported eIF4E-like genes on chromosome 4 and 20 (42), the fact that most of the fragments in Fig. 1A were cloned from chromosome 4-specific libraries strongly argues that the EIF4E1 gene is on chromosome 4. The entire length of cloned DNA comprising the EIF4E1 gene was 50 kb. The EIF4E2 gene was flanked by three Alu sequences and two 14-bp direct repeats and contained a 3'-terminal poly(A) stretch (Fig. 2). All of these features are characteristic of retrotransposons (43). Additional screening yielded DNA fragments representing intronless genes with more substantial differences from the cDNA and containing additional in-frame stop codons.


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Fig. 1.   Recombinant lambda  phage containing fragments of two different human eIF4E genes. Open boxes represent introns, and filled boxes represent exons. Arabic numbers refer to specific exons. Restriction endonuclease sites are indicated as follows: H, HindIII; E, EcoRI; N, NotI; C, ClaI; X, XhoI; and S, Sau3AI. Not all sites are shown, only those demonstrated directly for the various DNA fragments. A, the EIF4E1 gene. A composite based on all phage inserts for EIF4E1 is shown at the bottom. B, the EIF4E2 gene.


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Fig. 2.   Alignment of the nucleotide sequences of EIF4E1 and EIF4E2. A, the EIF4E1 gene. B, the EIF4E2 gene. The entire sequence for EIF4E2 from -712 to +1894 is given (see "Experimental Procedures" for numbering system), but the introns have been omitted from the EIF4E1 sequence. Exon-exon junctions in EIF4E1 are marked by vertical lines. The proposed major transcriptional initiation sites of the two genes at -80 and -19 are indicated by arrows. The transcriptional initiator region (Inr), translational initiation codons, and polyadenylation signals are boxed. Other consensus motifs within the putative promoter region of EIF4E2 are underlined. Three Alu repeats in EIF4E2 are indicated in boldface, and a 14-bp direct repeat is doubly underlined. The sequence of a 2.7-kb portion of lambda 4E2 has been deposited in GenBankTM/EMBL Data Bank (accession number, M77222).

Intron-Exon Structure of EIF4E1-- Comparison of the sequence of EIF4E1 with that of the cDNA (35) indicated that the gene is organized into seven exons and six introns (Fig. 1A). The regions in and around the exons were sequenced (Fig. 2). Exon 1 is the smallest (37 bp) and exon 7 is the largest (1.3 kb) (Table II). The introns of this gene range from 1.2 to more than 10 kb and include only two of the three possible types (44), types 0 and 2 (Table III). All the exon/intron junction sequences conform to the GT/AG rule (44) (Table IV).

                              
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Table II
Exons of the EIF4E1 gene

                              
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Table III
Introns of the EIF4E1 gene

                              
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Table IV
Exon-intron boundaries of the EIF4E1 gene
Exon sequence is presented in uppercase letters and intron sequence in lowercase. Exon sequences are grouped into codons.

The exon structure of EIF4E1 can be compared with the recently published three-dimensional structure of eIF4E (11) with respect to the theory that protein-coding genes evolved by assembly of exons encoding functional domains (45-46). Results of mass spectrometry and x-ray diffraction suggested that amino acids 1-35 are disordered in the absence of other proteins. These are encoded by exons 1 and 2 (Table II) which may have been added after the evolution of a core cap-binding protein for the purpose of binding other proteins (see Introduction). Exon 3 encodes most of beta  strand 1 (S1), the 1-2 loop, beta  strand 2 (S2), and most of alpha  helix 1 (H1). These structurally contiguous elements make up a region of the polypeptide chain that constitutes one side of the protein and include one of the two conserved Trp residues that "sandwich" the 7-methylguanine moiety, Trp-56. Exon 4 encodes essentially only one element, S3. Exon 5 encodes the 3-4 loop, S4, and most of H2. This collection of elements contains the other conserved sandwich Trp residue, Trp-102, as well as Glu-103, which forms hydrogen bonds to the N-1 and amino groups of 7-methylguanine. A similar set of interactions occurs between m7GMP and the dipeptide Trp-Glu (47), suggesting that exon 5 alone may have been a primordial cap-binding protein. Exon 6 encodes S5, S6, and most of H3, completing the "floor" of the cap-binding pocket. This portion of the protein contains most of the residues that bind the diphosphate of m7GDP, Arg-157, Lys-162, and Trp-166. Finally, exon 7 encodes H3, S7, H4, and S8. These contiguous elements make up the other side of the eIF4E molecule. This portion of the protein does not contain any of the residues that contact m7GTP but does contain the phosphorylation site (Ser-209), suggesting that it may have been a late evolutionary development appended for regulation of eIF4E activity.

Comparison of the EIF4E1 and EIF4E2 Genes-- Sequence alignment of the exonic portions of the EIF4E1 gene with the entire EIF4E2 gene indicates no differences in the 13-nt region immediately upstream of the ATG, four single base differences in the 651-nt coding region, and five single base differences in the 980-nt 3'-untranslated region up to nt 1641 (Fig. 2). In the 3'-untranslated region of the EIF4E2 gene there is also a 10-nt insertion of T residues after nt 847 and the absence of a 9-nt stretch from nt 1238 to 1246. Upstream of nt -13 and downstream of nt 1641 there is no similarity between the two genes. As previously reported (36), the promoter of EIF4E1 lacks a canonical TATA box but includes two consensus sites for c-Myc. The putative promoter of EIF4E2 also lacks a consensus TATA box but, importantly, contains a consensus initiator region (Inr), TCATACC. A strong match to the Inr consensus sequence is commonly seen in TATA-less promoters (48) and may serve as a binding site for the YY1 protein (49). Other consensus motifs within this region include GATA1, AP1, AP4, GFI1, NF-kappa B, STAT, c-Myb, SRY, SREBP1, and HFH2 (Fig. 2). The EIF4E2 gene contains only two of the three polyadenylation signals present in the eIF4E cDNA (35).

Both genes encode proteins of 217 amino acids with only two amino acid differences, Glu versus Lys at position 19 and Arg versus Trp at position 61. The location of the first of these sites in the three-dimensional structure of the protein is not known, but the second occurs at the beginning of S1. Amino acid residues at positions 19 and 61 do not make contact with m7GDP. Also, neither falls into the group of amino acid residues previously shown, by site-directed mutagenesis, to be important for cap binding (50-54). Finally, neither Glu-19 nor Arg-61 is universally conserved in all eIF4E sequences. For these reasons, we predict that eIF4E-2 would be a functional protein.

Transcription Initiation Sites-- Previous studies have indicated that transcription for mammalian eIF4E mRNA begins between -7 and -27; (i) the cloned human eIF4E cDNA begins at -18, although since this is a C residue, the mRNA is more likely to begin at the G at -19 (35); (ii) the cloned mouse eIF4E cDNA begins at -19 (55); and (iii) primer extension of mRNA from one mouse and one human cell line indicates a major start site at -16 and minor start sites at -7, -24, and -27 (55). The putative promoter of EIF4E2 bears no similarity to that of EIF4E1 and hence would be unlikely to initiate transcription in the same region. To determine whether EIF4E2 was transcribed, we performed primer extension with a primer complementary to the 5'-coding region of eIF4E mRNA, which should therefore produce extension products from both EIF4E1 and EIF4E2 transcripts. Primer extension using human placental RNA and primer 4 (Table I) produced major bands corresponding to initiation sites at -80 and -76 as well as minor bands at -19 and -20 (Fig. 3A). Primer extension with RNAs isolated from several human cell lines produced similar results (Fig. 3B). HeLa cell RNA (H) gave an initiation site at -80, the same as placental RNA, but Wish cell RNA (W) also gave minor products corresponding to initiation of transcription at -56 and -19. Chang cell RNA (C) yielded much more product corresponding to -19, less product from -80, and a small amount of product corresponding to -46 and -56. Finally, K562 cell RNA (K) produced even less of the -80 product, more of the -46 product, and a great deal of the -19 product.


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Fig. 3.   Localization of transcription initiation sites of eIF4E genes by primer extension. A, primer extension was performed with total RNA from human placenta (P). Extension products corresponding to major (-80 and -76 nt) and minor (-20 and -19 nt) transcription initiation sites are indicated. The DNA sequence ladder (GATC) represents the sequence of the 5'-region of EIF4E2 determined with the the same primer as that used for primer extension. B, primer extension was performed as in A with total RNA from four different human cell lines: Wish (W), Chang (C), K562 (K), and HeLa (H).

To determine the origins of the various primer extension products, we performed S1 and RNase protection analysis using probes based on EIF4E1 and EIF4E2 sequences. S1 mapping with RNA from Wish cells using a probe spanning from -1000 to +25 of the EIF4E1 gene produced a cluster of fragments corresponding to protection from -11 to -14 (Fig. 4A). As the EIF4E1 and EIF4E2 genes have the same sequences immediately downstream of -13 (Fig. 2), fragments of this size could be derived from either EIF4E1 transcripts initiated in the region of -11 to -14 or EIF4E2 transcripts initiated at this point or upstream. This results does, however, indicate that there are no unspliced transcripts from EIF4E1 beginning upstream of this point. It is possible, however, that a transcript is initiated upstream of -11 to -14 in EIF4E1 and then spliced, with the 3' splice site being located at -11 to -14. To test this possibility, we performed Southern analysis of restriction fragments of lambda 4E1-H (Fig. 1A), which contains ~20 kb upstream of the coding region, using a probe consisting of the -80 primer extension product eluted from the polyacrylamide gel. However, no positive hybridization was observed, indicating the lack of an additional upstream exon (data not shown).


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Fig. 4.   S1 and RNase protection analysis of EIF4E1 and EIF4E2 transcripts. A, S1 nuclease analysis performed with no RNA (N) or total RNA (R) from Wish cells using a single-stranded DNA probe from the 5'-region of the EIF4E1 gene. The numbers refer to nucleotide positions (Fig. 2). C, T, A, and G are a sequencing ladder derived from EIF4E1. B, RNase protection performed with an RNA probe from the 5'-region of the EIF4E2 gene and human placental RNA. pTCEEC RNA was transcribed in vitro from a linearized plasmid containing eIF4E cDNA (38) and corresponds to an EIF4E1 transcript. Numbers on the left correspond to nucleotide positions (Fig. 2). Numbers on the right give the sizes in nucleotides of RNA markers (M).

We tested directly for transcripts from EIF4E2 by ribonuclease protection analysis of human placental RNA using a radiolabeled RNA probe from -113 to +184 of the EIF4E2 gene (Fig. 4B, lane 1). A control RNA, transcribed in vitro from the cloned eIF4E cDNA contained in plasmid pTCEEC (38), produced a protected fragment at -13 as expected (lane 2). Placental RNA produced a number of protected fragments, the major ones corresponding to the probe terminating at -80 and -13 (lane 3). The -80 fragment is likely to be the same as the -80 primer extension product (Fig. 3), suggesting that transcription from EIF4E2 begins at -80. The -13 protection fragment is likely to have been produced by EIF4E1 transcripts, since the sequence of EIF4E1 and EIF4E2 is common only downstream of -13.

To confirm expression of the EIF4E2 gene, we performed RT-PCR using primers 3-6 (Table I). Primer 3 was used to make eIF4E-specific cDNA from human placental RNA. The sequence from -80 to +25 of the EIF4E1 gene can be specifically amplified by PCR using primers 4 and 6, and the sequence from -66 to +25 of the EIF4E2 gene can be specifically amplified using primers 4 and 5 (Fig. 5A). A product was obtained with primers 4 and 5 (Fig. 5B, lane 3) but not with primers 4 and 6 (lane 6), indicating that an mRNA is present in placental RNA that is initiated at or upstream of the -66 position of EIF4E2 but not at or upstream of position -80 of EIF4E1. This experiment was repeated using the -80 primer extension product of Wish cell RNA as template instead of placental RNA (Fig. 5C). Primers 4 and 5 allowed synthesis of a DNA product of the correct size (lane 2) but primers 4 and 6 did not (lane 4), providing direct proof that the -80 primer extension product of Fig. 4 is derived from EIF4E2. Finally, the PCR product in Fig. 5C, lane 2, was purified from the agarose gels and sequenced. The sequence matched the 5'-region of the EIF4E2 gene (Fig. 2). All of these results are evidence that mRNA transcribed from position -80 of EIF4E2 is present in human cells.


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Fig. 5.   RT-PCR analysis of EIF4E1 and EIF4E2 transcripts. A, RT-PCR primer design and strategy. Open boxes represent the protein coding regions. The scale indicates nucleotide position numbers, utilizing the numbering system described under "Experimental Procedures." Oligonucleotide primers are described under Table I. B, PCR was performed using the indicated primers and templates consisting of human placental total RNA, reverse-transcribed with primer 3 (R), lambda 4E1-H (1), and lambda 4E2 (2). The products were separated on 2% agarose gels. M, DNA markers. C, PCR was performed as in B except that in the indicated lanes (P) the template was the primer extension product terminating at -80 from Wish cell RNA, excised from a sequencing gel similar to that shown in Fig. 3B. The products were separated on 6% polyacrylamide gels.

Additional evidence that these upstream sequences represent bona fide promoter regions came from an analysis of their chromatin structure. Micrococcal nuclease digestion products derived from the upstream regions of EIF4E1 and EIF4E2 were found to be heterogeneous by ligation-mediated PCR, despite the fact that bulk chromatin in the same digests gave rise to typical nucleosome-protected ladders (data not shown). This result is consistent with these regions being sites of nucleosome disruption, a hallmark of transcriptional regulatory regions (56).

The evidence presented here supports the view that both EIF4E1 and EIF4E2 are expressed at the level of mRNA. Two major primer extension products were detected, corresponding to transcription starts at -80 and -19 (Fig. 3). The data indicate that EIF4E1 is initiated at -19 and that EIF4E2 is initiated at -80. Other examples have been described in which both intron-containing and intronless genes code for the same protein (43, 57-59), and in at least two cases, both genes yield functional proteins (43, 59). It is unclear, however, why human cells would express two functional genes for eIF4E. The fact that the promoter of EIF4E1 contains c-Myc-binding elements (36) whereas that of EIF4E2 does not suggests that the former may be inducibly expressed, e.g. during rapid cell growth, while the latter may be consititutively expressed. The differences in the relative amounts of the -80 and -19 primer extension products in various cultured human cell lines (Fig. 3B) suggests that the two genes may be differentially transcribed in response to factors such as growth rate or cell lineage. The differential expression of EIF4E1 and EIF4E2 may also explain why EIF4E2 cDNA was not initially cloned (35); fibroblast and lymphocytes, the sources for mRNA used in the cloning of human eIF4E cDNA, may express EIF4E1 predominantly. Similarly, the cultured cells chosen for primer extension analysis in the previously published study (55) may express EIF4E1 predominantly.

    ACKNOWLEDGEMENTS

We thank Kerry Blanchard, David S. Gross, Brent C. Reed, and Robert L. Smith for valuable advice; Dequan Chen, Chris Duggan, Weinu Gan, and Christopher A. Bradley for providing human cell lines; and the LSUMC Center for Excellence in Cancer Research, Treatment, and Education for use of the PhosphorImager.

    FOOTNOTES

* This work was supported by NIGMS Grant 20818 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M77222.

Dagger Current address: National Biosciences, 3650 Annapolis Ln., Suite 120, Plymouth, MN 55447.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Louisiana State University Medical Center, 1501 Kings Hwy., Shreveport, LA 71130-3932. Tel.: 318-675-5156; Fax: 318-675-5180; E-mail: rrhoad{at}lsumc.edu.

1 The abbreviations used are: eIF, eukaryotic initiation factor; PCR, polymerase chain reaction; kb, kilobase pair; bp, base pair; nt, nucleotide; RT-PCR, reverse transcriptase-polymerase chain reaction; PIPES, 1,4-piperazinediethanesulfonic acid; Inr, initiator region.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results & Discussion
References

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