A eukaryotic initiation factor 5C is upregulated during metamorphosis in the cotton bollworm, Helicoverpa armigera
© Dong et al; licensee BioMed Central Ltd. 2009
Received: 03 October 2008
Accepted: 08 March 2009
Published: 08 March 2009
The orthologs of eukaryotic initiation factor 5C (eIF5C) are essential to the initiation of protein translation, and their regulation during development is not well known.
A cDNA encoding a polypeptide of 419 amino acids containing an N-terminal leucine zipper motif and a C-terminal eIF5C domain was cloned from metamorphic larvae of Helicoverpa armigera. It was subsequently named Ha-eIF5C. Quantitative real-time PCR (QRT-PCR) revealed a high expression of the mRNA of Ha-eIF5C in the head-thorax, integument, midgut, and fat body during metamorphosis. Immunohistochemistry suggested that Ha-eIF5C was distributed into both the cytoplasm and the nucleus in the midgut, fat body and integument. Ha-eIF5C expression was upregulated by 20-hydroxyecdysone (20E). Furthermore, the transcription of Ha-eIF5C was down regulated after silencing of ecdysteroid receptor (EcR) or Ultraspiracle protein (USP) by RNAi.
These results suggested that during metamorphosis of the cotton bollworm, Ha-eIF5C was upregulated by 20E through the EcR and USP transcription factors.
To holometabolous insects, molting is a common physiological process, whose life cycles are characterized by a series of molts. During their larval molts, the larvae progress from one instar to the next. Thereafter, pupation and eclosion ensue during their metamorphic molts. Increasing evidence indicates that some hormones and receptors may contribute to the complex developmental pathways associated with molting and metamorphosis. Many genes have been shown to be involved in molting or metamorphosis, such as the transcription factors ecdysteroid receptor (EcR), Ultraspiracle protein (USP), Hormone receptor 3 (HR3) and Broad complex , and the programmed cell death pathway genes . Some key regulatory genes have also been identified, such as E74B and E93 . However, very few genes downstream of Broad complex, E74B and E93 have been identified. Consequently, there is a dearth of available knowledge on the molecular mechanisms that lead to larval molt and metamorphosis. By conducting a research of the molting related genes, we may further understand the molecular mechanism of development and ecdysone regulation, and find the novel molecular targets to effectively control the pest.
Suppression subtractive hybridization (SSH) is a useful method for identifying differentially expressed genes during larval molting. Using the metamorphically committed larvae (6th-72, 96 and 120 h) as the tester and the feeding 5th instar larvae (5th-24 h) as the driver, we obtained an EST, which was similar to basic leucine zipper by BLASTX analysis . We designed primers based on this fragment to obtain the full-length cDNA and identified it as translation initiation factor 5C (eIF5C).
The regulation of translation plays an important role in the control of gene expression. In eukaryotes, translation regulation occurs primarily during the initial step, which is rate limiting under most circumstances . More and more evidence suggests that translation initiation factors (eIFs) are not only essential in the initiation of protein translation but also important in other life processes. Some eIFs are regulators of signaling pathways, such as eIF4A of Drosophila melanogaster, which functions as a negative regulator of Dpp/BMP (decapentaplagic/bone morphogenetic protein) signaling that mediates SMAD (mother against dpp) degradation . Eukaryotic initiation factor 6 selectively regulates Wnt signaling and β-catenin protein synthesis .
eIF5C is a phylogenetically conserved protein, which is said to contain an N-terminal leucine zipper motif and a C-terminal eIF5C domain. Our BLASTX results showed that homologs of eIF5C exist in various organisms, from Cryptococcus neoformans to Homo sapiens. BZAP45, the ortholog of eIF5C in humans, contributes to transcriptional control at the G1/S phase transition . In Rattus norvegicus, brain development-related molecule 2 (Bdm2) is a developmentally regulated gene, which is highly expressed in fetal rat brain . Wang et al.  showed that eIF5C was associated with the ribosome through an interaction with D. melanogaster ribosomal protein L5 (dRPL5), suggesting its possible role during protein synthesis in fruit flies. Given that there are no related functional reports to date, the information of eIF5C from other insects have been obtained from gene sequencing.
In this study, we cloned and characterized the eIF5C from the metamorphic larvae of H. armigera and designated it as Ha-eIF5C, which contains an N-terminal leucine zipper motif and a C-terminal eIF5C domain. The expression, distribution and characterization of Ha-eIF5C were studied by employing Quantitative real-time PCR (QRT-PCR), recombinant expression and immunoblotting analysis. Likewise, we also investigated the gene's hormonal regulation and its position in the 20E signal transduction pathway.
Gene cloning and sequence analysis of Ha-eIF5C
Identification of Ha-eIF5C
Recombinant expression and purification of Ha-eIF5C
Tissue distribution and expression patterns of Ha-eIF5C
Hormonal regulations on Ha-eIF5C
At the same time, the localization of Ha-eIF5C in the integument and fat body was detected by immunohistochemistry. During the molting 5th and wandering 6th-96 h (Metamorphic molting), a cascade of physiological processes occurred. These included the separation of the old cuticle from the underlying epidermis, followed by the secretion of a new cuticle beneath the old. Finally, a shedding of the old exoskeleton occurred (Fig. 9-E, F). It was obvious that Ha-eIF5C appears localized in both cytoplasm and nucleus in the epidermis and lipocyte.
In this work, we identify a 1675 bp full-length elF5C from H. armegera. This includes a 1260 bp open reading frame encoding a 419 amino-acid protein with a predicted molecular mass of 48 kDa. Protein alignments showed that Ha-eIF5C and eIF5C from A. aegypti, A. mellifera, D. melanogaster and B. mori are very similar.
Ha-eIF5C is a phylogenetically conserved protein predicted to contain an N-terminal leucine zipper motif (39–60 aa) and a C-terminal eIF5C domain (326–411 aa). This eIF5C domain was first detected at the very C-termini of the yeast protein GCD6, eIF-2B epsilon and two other eukaryotic translation initiation factors, eIF-4 gamma and eIF-5, and may likewise be involved in the interaction of eIF-2B, eIF-4 gamma and eIF-5 with eIF-2 . Therefore, this eIF5C domain in Ha-eIF5C implies that Ha-eIF5C might also function as a novel translation initiation factor.
Leucine zipper motifs are protein-protein dimerization motifs consisting of heptad repeats of leucine residues that form a coiled-coil structure . These motifs have been well described in the context of transcription factors such as c-Fos and c-Jun, where they mediate homo- and hetero-dimerization critical for the DNA binding properties of these transcription factors . Proteins containing leucine zipper motif have been reported to be related with larval growth, molting and metamorphosis in D. melanogaster [14, 8]. Our work provides evidence that the expression of Ha-eIF5C, which contains a leucine zipper motif, is upregulated during metamorphosis. We speculate that through its leucine zipper domain, Ha-eIF5C may be involved in transcriptional regulation during insect development.
The expression profile of Ha-eIF5C was correlated with the metamorphic process of H. armigera. In our study, Ha-eIF5C was upregulated during metamorphosis in the head-thorax, integument, midgut and fat body. Thummel  reported that apoptotic and autophagic programmed cell death pathways are involved in tissue histolysis and remodeling during metamorphosis. Gorski et al.  confirmed that programmed cell death in the salivary glands of D. melanogaster requires active protein synthesis, even though cell death is a degradative cellular process. Gorski et al.  also found significant upregulation of several translation-initiation factors.
Molting and metamorphosis of larvae are very important physiological behaviors in insects, and are governed by two hormones, namely, 20E and juvenile hormone (JH) . Wang et al.  showed that H. armigera had a similar developmental schedule as compared with Manduca sexta. 20E levels increase during the late stages of the final (wandering) instar in M. sexta larvae, before pupal ecdysis, and then decrease at the pupal ecdysis . The expression of the Ha-eIF5C transcript went with the titer of 20E and was enhanced after being injected with 20E, which suggested that it was regulated by 20E in vivo. Moreover, the fact that the expression level of Ha-eIF5C in HaEpi cell line decreased after EcR or USP was knocked down demonstrated that Ha-eIF5C was upregulated by 20E via EcR or USP transcription factor.
Ha-eIF5C appears localized in both the cytoplasm and the nucleus in the midgut, integument and fat body. However, it was identified as a cytoplasmic protein in D. melanogaster . In light of its function in the initiation phase of protein synthesis, eIFs were often targeted in the cytoplasm. However, some eIFs such as eIF4E are distributed in the cytoplasm and the nucleus. In the cytoplasm, eIF4E acts in the rate-limiting step of translation initiation. In the nucleus, eIF4E facilitates the nuclear export of a subset of mRNAs. Both of these functions contribute to eIF4E's ability to oncogenically transform cells . Neither eIF5C nor eIF4E contain classical nuclear localization signals (NLSs) predicted by the bioinformatics method http://cubic.bioc.columbia.edu/cgi/var/nair/resonline.pl. They might act in consonance with some assistant factors that are imported into the nucleus. Dostie et al.  demonstrated that eIF4E-Transporter (4E-T) is a nucleocytoplasmic shuttling protein that contains an eIF4E-binding site, one bipartite NLS and two leucine-rich nuclear export signals, which mediate the nuclear import of eIF4E via the importin αβ pathway by a piggy-back mechanism.
Ha-eIF5C possibly functions as a novel translation initiation factor in protein synthesis just like eIF5C of D. melanogaster. However, it was interesting to find that Ha-eIF5C was upregulated during metamorphosis. Likewise, it was equally interesting to discover that the expression of Ha-eIF5C transcript was enhanced by 20E through EcR and USP. Thus, we hypothesize that Ha-eIF5C possibly functions as a regulator of cotton bollworm development, in addition to its role as a translation initiation factor.
The larvae of the cotton bollworm were maintained in this laboratory with an artificial diet described by Zhao et al. at 28°C under a light:dark ratio of 14:10 h . Moths were fed with 2% sugar water.
Molecular cloning of Ha-eIF5C gene
A fragment of Ha-eIF5C was obtained by suppression subtractive hybridization (SSH) using the metamorphically committed larvae (6th-72, 96 and 120 h) as the tester and the feeding 5th instar larvae (5th-24 h) as the driver . The full-length cDNA was cloned using the cDNA library of H. armigera as a template. The 3' end of the gene was amplified using a gene-specific forward primer, eIF5CF (5'-aactccagcaagggcaagatg-3') and a T7 primer (5'-taatacgactcactataggg-3'). Similarly, the 5' end of the cDNA was amplified by a T3 primer (5'-aattaaccctcactaaaggg-3') and a reverse gene-specific primer eIF5CR (5'-tcttcttcggcgctctgtagc-3').
Similarity analysis was performed by BLASTX http://www.ncbi.nlm.nih.gov/. Gene translation and prediction of the deduced protein were performed by ExPASy Proteomics Server http://www.expasy.ch/tools/, including compute pI/Mw, TMpred, NetPhos, NetNGlyc and NetOGlyc. Signal sequence and motif prediction utilized SMART http://smart.embl-heidelberg.de/. Alignments were performed with ClustalW http://www.ebi.ac.uk/clustalw/index.html and GENDOC computer programs http://www.psc.edu/biomed/genedoc/.
Recombinant expression and purification
A pair of primers (eIF5CexpF: tactcaggatccatgagtcagaaggtagaaaaac; eIF5CexpR: tactcagtcgacctaatcttcttcttcgccactc) were designed to amplify the sequence coding for Ha-eIF5C protein (bold indicates BamH I and Sal I sites, respectively). The DNA fragment was cut with BamH I and Sal I, ligated into expression vector pGEX-4T-1 and transformed into competent Escherichia coli BL21 host cells. The recombinant expression of Ha-eIF5C was induced by 0.1 mM Isopropyl β-D-1-Thiogalactopyranoside (IPTG). Thereafter, the cells were centrifuged (6000 g, 10 min), resuspended with Phosphate-Buffered Saline (PBS, 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4) containing 0.1% Triton X-100 and sonicated. The recombinant GST-eIF5C was expressed in supernatant and purified by Glutathione Sepharose 4B.
Rabbit polyclonal antiserum against Ha-eIF5C was prepared using recombinant protein purified from E. coli by SDS-PAGE. About 200 μg protein was diluted with saline and mixed with the same volume of complete Freund's adjuvant. It was then injected hypodermically into the back of the rabbit. After three weeks, the emulsified mixture of 200 μg purified recombinant protein and incomplete Freund's adjuvant was then subcutaneously injected into the rabbit. Two weeks later, the rabbit was given booster injections of 500 μg antigen without adjuvant and the antiserum samples were collected. The specificity of the antiserum was examined by immunoblotting and the antiserum was used in all the immunoassay experiments.
We followed previously reported procedures . Protein extracts (100 μg) of the H. armigera tissues were separated using 12.5% SDS-PAGE and transferred onto a nitrocellulose membrane. Antiserum against Ha-eIF5C was diluted 1:100 in 2% non-fat milk in Tris-buffered saline (TBS, 10 mM Tris-HCl, pH 7.5 and 150 mM NaCl) and the second antibody of Horseradish Peroxidase (HRP) conjugated to goat anti-rabbit IgG was diluted 1:10,000 in the same blocking buffer (2% non-fat milk in TBS).
Quantitative real-time PCR analysis
Total RNA was isolated from the head-thorax, integument, midgut, fat body and haemocytes at different developmental stages using Unizol reagent according to the manufacturer' s protocol (Biostar, Shanghai, China). A total of 5 μg RNA was used to reverse transcribe the first strand cDNA (First Strand cDNA Synthesis Kit, MBI Fermentas, St. Leon-Rot, Germany). It was subsequently used as a template in the PCR reactions.
SYBR green-based quantitative real-time PCR (Q-PCR) analysis was performed using PTC-200 DNA Engine thermal cycler (MJ Research) and chromo4 four-color real-time detector (Bio-Rad, America). The following primers were used to amplify a specific fragment of 102 bp: eIF5CF1 (5'-tatggcaatgtgtgatgtcccgtg-3'); eIF5CR1 (5'-cagccaacagcggcgtgtaatg-3'). A 150 bp fragment of β-actin was also amplified as control, with the primers actinF (5'-cctggtattgctgaccgtatgc-3') and actinR (5'-ctgttggaaggtggagagggaa-3'). Amplification conditions were 95°C, 2 min; 40 cycles (95°C, 15 s; 62°C, 50 s; incubated at 72°C for 2 s; plate read; incubated at 82°C for 2 s; plate read); melting curve from 60°C to 95°C, read every 0.5°C, hold 1 s. The data provided from real-time PCR instrumentation were then prepared for input into Microsoft Excel and analyzed using the 2-ΔCT method .
Hormonal regulation of Ha-eIF5C
The 6th instar 0 h larvae (6th-0 h) were injected with steroid 20E (500 ng/larva). 20E was first dissolved to 10 mg/ml in dimethyl sulphoxide (DMSO) and then diluted into 0.1 mg/ml with PBS when injecting worms. Untreated controls were only injected by equivalent amounts of carrier. Total RNA of the midgut was extracted from the injected worms at different developmental periods. A comparison of the differences between the control and the challenged was done by RT-PCR with gene specific primers: eIF5CF1 (5'-tatggcaatgtgtgatgtcccgtg-3'); eIF5CR (5'-tcttcttcggcgctctgtagc-3'). The following procedure was employed: one cycle (94°C, 2 min); 26 cycles (94°C, 30 s; 53°C, 45 s; 72°C, 45 s), followed by a last cycle (72°C, 10 min). The β-actin gene was used for normalization. Each experiment was repeated three times independently. Ratios of Ha-eIF5C to β-actin were calculated with Quantity One (Bio-Rad, Hercules, CA, USA).
The primers of EcRRNAiF1 (5'-gcgtaatacgactcactataggcgctggtataacaacggagga-3') and EcRRNAiR1 (5'-gcgtaatacgactcactataggagctggagacaactcctcacg-3'), EcRRNAiF2 (5'-cgctggtataacaacggagga-3') and EcRRNAiR2 (5'-agctggagacaactcctcacg-3'), USPRNAiF1 (5'-gcgtaatacgactcactataggcgaaccatcccctaagtggttc-3') and USPRNAiR1 (5'-gcgtaatacgactcactataggccttgatgagcaggatctggtc-3'); USPRNAiF2 (5'-cgaaccatcccctaagtggttc-3') and USPRNAiR2 (5'-ccttgatgagcaggatctggtc-3'); GFPRNAiF1 (5'-gcgtaatacgactcactataggtggtcccaattctcgtggaac-3') and GFPRNAiR1 (5'-gcgtaatacgactcactataggcttgaagttgaccttgatgcc-3'); GFPRNAiF2 (5'-tggtcccaattctcgtggaac-3') and GFPRNAiR2 (5'-cttgaagttgaccttgatgcc-3') were used for PCR to amplify the gene fragments. PCR products were purified using a PCR purification kit. dsRNA was synthesized using the MEGAscript™ RNAi kit (Ambion Inc, Ausdin, USA). The procedures of culturing HaEpi cell line and RNAi were performed according to Shao et al. . The green fluorescence protein (GFP) was used as control.
The midguts and integuments adhering with fat bodies were dissected in PBS and fixed for 10 h in 4% paraformaldehyde at 4°C. The tissues were dehydrated with a graded series of ethanol. Protein digestion was performed by incubating with proteinase K (20 μg/ml) for 15 min at 37°C. Sections were blocked in 2% bovine serum albumin (BSA), incubated with a primary antibody against Ha-eIF5C diluted to 1:100, and then with a goat anti-rabbit-ALEXA 488 antibody (Eugene, United States) diluted to 1:1000 in PBS with 2% BSA at room temperature for 30 min. The nuclei were stained with 4'-6-Diamidino-2-phenylindole dihydrochloride (DAPI, 1 μg/mL in water, San Jose, United States) for 10 min. Negative controls were treated in the same manner, but pre-immune rabbit serum was used in place of the antiserum against Ha-eIF5C. Fluorescence was detected with an Olympus BX51 fluorescence microscope.
The nucleotide sequence reported in this paper has been submitted to GenBank with accession number [GenBank: EU526835].
This work was supported by grants from the National Basic Research Program of China (2006CB102001) and the National Natural Science Foundation of China (No: 30710103901, 30670265).
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