From the Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport, Louisiana 71130-3932
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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EXPERIMENTAL PROCEDURES |
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Materials--
Two human placental genomic libraries in
bacteriophage vectors (EMBL3-SP6/T7 and
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
[-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.
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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 [-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
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 [
-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
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.
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RESULTS AND DISCUSSION |
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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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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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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-
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).
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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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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