From the § Department of Biochemistry and Molecular
Biology, Louisiana State University Medical Center, Shreveport,
Louisiana 71130-3932 and the Departments of Chemistry and
¶ Biophysics, University of Warsaw,
02-093 Warsaw, Poland
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ABSTRACT |
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The rate-limiting step for cap-dependent translation initiation in eukaryotes is recruitment of mRNA to the ribosome. An early event in this process is recognition of the m7GTP-containing cap structure at the 5'-end of the mRNA by initiation factor eIF4E. In the nematode Caenorhabditis elegans, mRNAs from 70% of the genes contain a different cap structure, m32,2,7GTP. This cap structure is poorly recognized by mammalian elF4E, suggesting that C. elegans may possess a specialized form of elF4E that can recognize m32,2,7GTP. Analysis of the C. elegans genomic sequence data base revealed the presence of three elF4E-like genes, here named ife-1, ife-2, and ife-3. cDNAs for these three eIF4E isoforms were cloned and sequenced. Isoform-specific antibodies were prepared from synthetic peptides based on nonhomologous regions of the three proteins. All three eIF4E isoforms were detected in extracts of C. elegans and were retained on m7GTP-Sepharose. One eIF4E isoform, IFE-1, was also retained on m32,2,7GTP-Sepharose. Furthermore, binding of IFE-1 and IFE-2 to m7GTP-Sepharose was inhibited by m32,2,7GTP. These results suggest that IFE-1 and IFE-2 bind both m7GTP- and m32,2,7GTP-containing mRNA cap structures, although with different affinities. In conjunction with IFE-3, these eIF4E isoforms would permit cap-dependent recruitment of all C. elegans mRNAs to the ribosome.
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INTRODUCTION |
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All eukaryotic cytosolic mRNAs and many eukaryotic viral mRNAs contain a 5'-terminal capping group (1). The most commonly occurring cap structures contain 7-methylguanosine in a 5'-to-5' triphosphate linkage to the first transcribed nucleotide residue, which is often 2'-O-methylated as well. The presence of a cap on mRNA stimulates translation as well as stabilizes the mRNA against degradation. The former of these effects is thought to be mediated by the binding of a 25-kDa initiation factor, eIF4E (eukaryotic initiation factor 4E), to the cap (2). eIF4E is a member of the eIF4 class of initiation factors, which also includes eIF4A, eIF4B, and eIF4G; collectively these factors recruit mRNA to the 43 S initiation complex and melt mRNA secondary structure (3). The primary structure of eIF4E has been deduced from cDNA in a variety of species (4-9), and the tertiary structure has recently been solved in the case of mouse (10) and yeast (11). In plants, there are at least two eIF4E isoforms, termed eIF4E and eIF(iso)4E (12, 13); the former is expressed in most tissues, whereas the latter is expressed only in floral organs and developing tissues (9).
eIF4E 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 (4) and increases the affinity of the protein for cap analogues 3-4-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 (15). Third, eIF4E levels are regulated at the transcriptional level (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, transformation in culture and tumorigenesis in nude mice, prevention of apoptosis in growth factor-restricted fibroblasts, 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-2 (reviewed in Ref. 17). Reduction in intracellular eIF4E levels by expression of antisense RNA results in phenotypic reversal of ras-transformed fibroblasts (18). 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 (19), and naturally occurring breast and head-and-neck tumors express elevated levels of eIF4E (Ref. 20 and references therein).
The structural requirements for recognition of the cap by mammalian and plant eIF4E have been determined by inhibition of in vitro translation by cap analogs, quenching of tryptophan fluorescence in eIF4E by cap analogs, and translation of mRNAs synthesized with modified cap structures (reviewed in Ref. 2). Binding requires the presence of the 7-methyl group, but changing the substituent at N7 to an aromatic group increases binding affinity. Addition of one methyl group at the N2 position of guanine has little effect on binding, but addition of a second methyl group (m32,2,7GTP; Scheme 1) drastically decreases it (21-23), presumably due to the loss of the H-bond between the N2 of m7G and Glu-103 of eIF4E (10).
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Most nematode mRNAs have a 22-nucleotide trans-spliced leader sequence at their 5'-ends (24, 25). In Caenorhabditis elegans, a number of different spliced leaders have been described (26), and mRNAs from ~70% of the genes contain a spliced leader (27). Trans-splicing results in an mRNA with an m32,2,7GTP-containing cap structure (28, 29). Although the effect of the trimethyl cap on translational efficiency in C. elegans has not been studied, trans-splicing enhances the translational efficiency of mRNAs in the parasitic nematode Ascaris (25). This increase reflects synergistic effects of both the spliced leader and the m32,2,7GTP-containing cap.
The inability of eIF4E from the species studied to date to bind m32,2,7GTP-containing caps contrasts with the fact that most mRNAs of C. elegans contain such caps, suggesting that eIF4E from C. elegans may differ qualitatively from that of other species. We therefore set out to isolate eIF4E from C. elegans to determine its properties. Surprisingly, we found multiple eIF4E isoforms. Moreover, the different types varied in their abilities to recognize m7GTP and m32,2,7GTP.
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EXPERIMENTAL PROCEDURES |
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Materials-- Carbenicillin, leupeptin, pepstatin, TAME,1 and aprotinin were purchased from Sigma. Maleimide-activated keyhole limpet hemacyanin and bovine serum albumin were purchased from Pierce. Ni2+-nitrilotriacetic acid-agarose was obtained from Qiagen (Chatsworth, CA). The protease inhibitor E-64 and CompleteTM Protease Inhibitor tablets (without EDTA) were obtained from Boehringer Mannheim. Affi-Gel 501 resin was obtained from Bio-Rad. The following peptides (derived from C. elegans eIF4E sequences but with an additional N-terminal Cys residue) were synthesized by Biosynthesis (Dallas, TX) and used to generate isoform-specific antibodies for IFE-1, -2, and -3, respectively: CLSLHSSDAPVAEKS, CKHAIYAVEPREEK, and CPRICLPAKDPAPVK. The following oligodeoxynucleotides were synthesized by Life Technologies, Inc. and termed Primers 1-8, respectively: CGAACCGGATCCATGACTGAAACGGA, GAGGGACGCAAGCTTAGACGGCGGATTTCTCG, TCCGAGGATCCAGTCGCAGCTCC, AAGAAAGCTTAGTGGCTGGTGTGGCAGG, GAAACCATGGGCACATCCGTAGCGGAA, TGGTGAGATCTCGAGAATATGCTTAAGGAG, CTGCCAGAAGAAGAAGACCAGCTC, and CCATCTCGAGAGCTGGCAACCAC. The plasmid pET32A and the S-Protein:bacterial alkaline phosphatase conjugate were purchased from Novagen (Madison, WI). DNA sequencing was performed at the Iowa State University Sequencing Facility.
Synthesis of Cap Analogs and Affinity
Resins--
m7GTP was synthesized by methylation of GTP
(30). m32,2,7GTP was obtained in a multi-step
synthesis from 5-amino-1--D
ribofuranosyl-4-imidazolecarboxamide (31). The affinity chromatography
resins m7GTP-Sepharose and
m32,2,7GTP-Sepharose were synthesized as
described previously (32) with modifications (31).
Preparation of C. elegans Extracts--
C. elegans
wild type strain N2 var. Bristol was cultured (33) and maintained on
Petri plates containing nematode growth medium agar and
Escherichia coli strain OP50 (34). Large quantities of
C. elegans were grown on plates supplemented with chicken
egg yolk (35). Animals were harvested, cleaned by one or more rounds of
sucrose flotation (34), pelleted, resuspended in an equal volume of
water, and drop-frozen in liquid N2. Frozen tissue was crushed with a mortar and pestle under liquid N2 and
thawed in the presence of buffer components and inhibitors to achieve
the following final concentrations: 20 mM MOPS, pH 7.5, 1 mM EDTA, 2 mM EGTA, 100 mM KCl, 0.5 mM dithiothreitol, 80 µg/ml each leupeptin and pepstatin,
10 µg/ml E-64, 1 mg/ml TAME, 50 mM NaF, and 10 mM -glycerophosphate. Homogenates were centrifuged at
20,000 × g for 15 min at 4 °C, and the supernatant
solutions were immediately applied to affinity chromatography
columns.
Affinity Chromatography-- C. elegans extracts (10-20 ml) were applied to 0.2-ml columns of m7GTP-Sepharose or m32,2,7GTP-Sepharose equilibrated in buffer A (20 mM MOPS, pH 7.5, 1 mM EDTA, 100 mM KCl, 10% (v/v) glycerol, and 0.5 mM dithiothreitol), and the flow-through fraction was collected. Columns were washed with 10 ml of buffer A followed by 10 ml of buffer A containing 100 µM GTP. Proteins were eluted with 2 ml of buffer A containing either 100 µM m7GTP or m32,2,7GTP (depending on the column matrix), and 0.2-ml fractions were collected.
Sequences of eIF4E Genes and cDNAs from C. elegans-- All of the predicted protein sequences were first identified in the genomic sequences generated by the C. elegans Genome Sequencing Consortium (36). The TBLASTN algorithm (37) run on the Washington University Genome Sequencing Center server2 was used to identify sequences that encode proteins with homology to human eIF4E. Expressed sequence tags from each of the C. elegans eIF4Es were identified with the C. elegans EST data base BLAST server at the DNA Data Bank of Japan.3
Cloning of C. elegans eIF4E cDNAs--
The coding sequences
of IFE-1, IFE-2, and IFE-3 were amplified from total C. elegans cDNA (35) by PCR using Primers 1 and 2, 3 and 4, or 5 and 6, respectively. The products were subcloned into the
BamHI/HindIII,
BglII/HindIII, or NcoI/XhoI
sites of pET32A to generate the vectors pTSIFE-1, pTSIFE-2, and
pTSIFE-3, respectively. The last 48 bp of the IFE-1 coding region were
lost during subcloning due to the presence of a HindIII site
located upstream of the 3' PCR primer site. Because this region of the
gene does not contain intron sequences, genomic DNA was used as
template for amplification of this portion of the coding sequence using
Primers 7 and 8. The resulting product, containing the missing 48 bp
plus an additional 752 bp, was subcloned into the
HindIII/XhoI sites of pTSIFE-1 to create
pTSIFE-1+. The constructs expressed eIF4E isoforms containing an
N-terminal addition consisting of thioredoxin, an S-peptide sequence,
and a His6-tag. C. elegans eIF4E cDNAs were
also kindly provided by the Yuji Kuhara laboratory (Japan) as Zap
clones corresponding to expressed sequence tags for IFE-1 (yk364a1), IFE-2 (yk452e8), and IFE-3 (yk81f11). All cDNA constructs were sequenced and compared with genomic sequences to determine intron/exon boundaries. Two discrepancies were observed. First, a 9-nucleotide insertion was present in the IFE-3 coding region of yk81f11 but not
pTSIFE-3, resulting in the addition of Lys-Leu-Gln between Gln-115 and
Arg-116; this may represent an alternatively spliced form of IFE-3
mRNA. Second, a single nucleotide change was observed in pTSIFE-2
compared with yk452e8 and genomic DNA, resulting in Pro-114 instead of
Leu-114. This likely represents a PCR-induced mutation.
Expression and Purification of Recombinant
Proteins--
Expression of recombinant C. elegans eIF4E
isoforms was induced with 1 mM
isopropyl-1-thio--D-galactopyranoside for 3 h in 1-liter cultures of E. coli strain BL21(DE3)pLysS (38)
bearing plasmids pTSIFE-1+, pTSIFE-2, or pTSIFE-3. Cells were cooled in an ice water bath, pelleted by centrifugation, and stored at
70 °C. Cells were thawed in the presence of buffer B (25 mM Tris·HCl, pH 7.5, 300 mM NaCl, 5 mM
-mercaptoethanol, and one CompleteTM Protease
Inhibitor tablet/25 ml) and lysed by sonication (3-6 bursts of 10 s each). The 28,000 × g supernatant was incubated with
300 µl of Ni2+-nitrilotriacetic acid-agarose with slow
rotation for 2 h at 4 °C. The resin was washed with buffer B
until the A280 nm of the supernatant was below
0.01. The resin was then washed with 10 ml of buffer B containing 40 mM imidazole, and fusion proteins were eluted with five
column volumes of buffer B containing 100 mM imidazole.
Immunological Procedures-- Preparation of anti-peptide antibodies and immunoblotting were performed as described previously (39). Antibodies were purified on columns of Affi-Gel 501 to which each synthetic peptide was linked via the Cys residue (40).
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RESULTS AND DISCUSSION |
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m7GTP- and m32,2,7GTP-Sepharose affinity chromatography resins were synthesized to determine whether eIF4E from C. elegans recognizes mono- or trimethylated cap structures. Extracts from C. elegans were prepared under conditions that in other systems preserve cap binding activity and minimize proteolysis and dephosphorylation of proteins. The eluate from the m7GTP-Sepharose column consisted of a complicated pattern of proteins ranging from ~20 to 200 kDa, the most intensely staining of which migrated between 26 and 40 kDa (Fig. 1A, lanes 5-7). These bands represent proteins that were specifically retained on m7GTP-Sepharose, because they were not eluted by the GTP wash (Fig. 1A, lane 4). This complex pattern of proteins is similar to that observed with extracts from higher eukaryotes (Ref. 41 and references therein), for which it has been shown that the major band represents eIF4E, whereas the others represent initiation factors that specifically associate with eIF4E (i.e. eIF4G, eIF4A, eIF3, eIF4B, and eIF4E-binding proteins). Chromatography on m32,2,7GTP-Sepharose produced a simpler collection of retained proteins, with bands at 26 and 37 kDa predominating (Fig. 1B, lanes 5-7). Inclusion of GTP in the extract reduced the amount of the 37-kDa band relative to the 26-kDa band (Fig. 1C), suggesting that the former protein is nonspecifically retained. The 26-kDa band from m7GTP-Sepharose co-migrated with the major protein retained on m32,2,7GTP-Sepharose (data not shown). The slower migrating proteins (Fig. 1, B and C) appear to correspond in molecular mass to a subset of the proteins retained on m7GTP-Sepharose (Fig. 1A) and are presumed to be eIF4E-associated initiation factors. These results indicate, based on molecular mass and retention on affinity columns, that there are several candidates for C. elegans eIF4E.
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Identification of eIF4E cDNA and Gene Sequences in Genomic Data Bases-- Three genes (hereby named ife-1, ife-2, and ife-3) that encode proteins with strong homology to human eIF4E were identified in sequences generated by the C. elegans Genome Sequencing Consortium (36). cDNAs corresponding to each of the C. elegans ife genes were cloned and sequenced. The calculated molecular masses of the predicted eIF4E proteins, IFE-1, IFE-2, and IFE-3 (Fig. 2A), were 24.3, 25.7, and 27.8 kDa, respectively. Each of the IFE proteins showed strong homology to human eIF4E and to each other (Fig. 2B).
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Development of Immunological Reagents-- Specific antisera were generated to distinguish between the various eIF4E isoforms. Peptides corresponding to sequences located in the nonhomologous C-terminal portion of the proteins (Fig. 2A, boldface) were synthesized and used for generation and purification of antibodies. To test the specificity of the antibodies, the cDNAs for IFE-1, IFE-2, and IFE-3 were cloned into a bacterial vector and expressed as fusion proteins (rIFE-1, -2, and -3). When the mass of the N-terminal extension is taken into account, these recombinant proteins migrated on SDS-polyacrylamide gel electrophoresis as expected (Fig. 3A). Each anti-peptide antibody recognized only the corresponding isoform of eIF4E when tested against the purified recombinant proteins (Fig. 3, B-D). The protein band migrating below rIFE-2 (arrow) likely represents a truncated form of rIFE-2 because it contains the N-terminal S-peptide (data not shown) but lacks the C-terminal IFE-2 epitope.
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ACKNOWLEDGEMENTS |
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We are grateful to Yuji Kuhara for providing C. elegans cDNAs, Kim Depper for growing and harvesting C. elegans, and the C. elegans Genome Sequencing Consortium for making genomic sequences available.
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Note Added in Proof |
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The Genome Sequencing Consortium has identified two additional eIF4E-like genes in C. elegans: ife-4, identified as predicted gene C05D9.5, and ife-5, located on YAC Y57A10, suggesting an even greater degree of complexity of regulation of translation and/or development in C. elegans.
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FOOTNOTES |
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* This work was supported by Grant GM20818 from the National Institute of General Medical Sciences, Grant MEN/NSF-96-257 from the U.S.-Poland Maria Sklodowska-Curie Joint Fund II, Grant 6 P04A 03409 from The Polish Committee for Scientific Research, and Grant RR10296 from the National Center of Research Resources.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.
To whom correspondence should be addressed: Dept. of
Biochemistry and Molecular Biology, Louisiana State University Medical Center, 1501 Kings Highway, Shreveport, LA 71130-3932. Fax:
318-675-5180; E-mail: rrhoad{at}lsumc.edu.
1
The abbreviations used are: TAME,
N-p-tosyl-L-arginine methyl ester; MOPS,
3-(N-morpholino)propanesulfonic acid; PCR, polymerase chain
reaction; bp, base pairs.
2 The website is www.genome.wustl.edu/gsc/blast/blast_servers.html.
3 The website is www.ddbj.nig.ac.jp/c-elegans/html/CE_BLAST.html.
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REFERENCES |
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