Eur J Biochem 271, 2923–2936 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04217.x Two functionally redundant isoforms of Drosophila melanogaster eukaryotic initiation factor 4B are involved in cap-dependent translation, cell survival, and proliferation ´ ´ ´ Greco Hernandez1, Paula Vazquez-Pianzola1, Andreas Zurbriggen2, Michael Altmann2, Jose M Sierra3 and Rolando Rivera-Pomar1 Max-Planck-Institute fuăr biophysikalische Chemie, Goăttingen, Germany; 2Institut fuăr Biochemie und Molekularbiologie, Universitaăt Bern, Switzerland; 3Centro de Biologı´a Molecular ‘Severo Ochoa’, Universidad Auto´noma de Madrid, Spain Eukaryotic initiation factor (eIF) 4B is part of the protein complex involved in the recognition and binding of mRNA to the ribosome Drosophila eIF4B is a single-copy gene that encodes two isoforms, termed eIF4B-L (52.2 kDa) and eIF4B-S (44.2 kDa), generated as a result of the alternative recognition of two polyadeynlation signals during transcription termination and subsequent alternative splicing of the two pre-mRNAs Both eIF4B mRNAs and proteins are expressed during the entire embryogenesis and life cycle The proteins are cytoplasmic with polarized distribution The two isoforms bind RNA with the same affinity eIF4B-L and eIF4B-S preferentially enhance cap-dependent over IRES-dependent translation initiation in a Drosophila cellfree translation system RNA interference experiments suggest that eIF4B is required for cell survival, although only a modest reduction in rate of protein synthesis is observed Overexpression of eIF4B in Drosophila cells in culture and in developing eye imaginal discs promotes cell proliferation The control of mRNA translation is a central process in the regulation of gene expression Regulation of mRNA translation preferentially takes place at the initiation level, and mRNA binding to the ribosome is a rate-limiting step in translation initiation [1] Translation initiation begins with the recognition of the 5¢-UTR of an mRNA by proteins that catalyze the landing of 40S ribosomes such as the eukaryotic initiation factor (eIF)4F complex, which is composed of factors eIF4E, eIF4A and eIF4G [2] Other initiation factors such as eIF1, eIF1A, eIF4B, and eIF5 are also required eIF4E recognizes the cap structure (m7GpppN) at the 5¢-UTR end of cellular mRNAs eIF4A is an ATPase/RNA helicase which is thought to unwind secondary structure at the 5¢-UTR of mRNAs to help 40S ribosomes to scan to the initiation codon eIF4G is a scaffold/adaptor protein which binds to the cap-binding protein eIF4E, as well as to eIF4A and to further factors such as poly(A)-binding protein (PABP) and ribosome-associated eIF3 [2] For picornaviral mRNAs and some cellular mRNAs, 5¢-UTR recognition occurs independently of the cap structure and is mediated by an internal ribosome entry site (IRES) located in proximity to the initiation codon [3] eIF4B was identified, and its genes were cloned from mammals [4,5], yeast [6,7], and wheat [8,9] The role of eIF4B in the initiation of translation is not well understood Its main function is assumed to be in the scanning process because eIF4B stimulates ATP-dependent unwinding of the mRNA 5¢-UTR by eIF4F/eIF4A eIF4B transiently associates with eIF4F [4,10,11] and stimulates the ATPdependent RNA-helicase activity of eIF4A and eIF4F in vitro [7,10,12–15] eIF4B is able to anneal complementary RNA strands in the absence of eIF4A [16,17] It binds nonspecifically to RNA via an RNA recognition motif (RRM) located at the N-terminus and also via a basic amino-acid sequence located at its C-terminus [5–7,11, 16–18] There is also evidence that eIF4B might facilitate binding of 40S ribosomes to the mRNA The RRM of mammalian eIF4B specifically binds to 18S rRNA [19] The central part of the mammalian protein contains a region rich in Asp, Arg, Tyr and Gly (DRYG), which, in addition to mediating homodimerization, is required for its interaction with eIF3 [20] The N-terminus of human eIF4B mediates the interaction with PABP [21] Genetic evidence [6,22] and in vitro experiments [23,24] support the model that eIF4B bridges eIF3 to the 40S subunit Additional experiments suggest that mammalian eIF4B binds to picornaviral IRESs and is involved in IRES-dependent translation of these mRNAs, although this interaction is apparently not essential for IRES-dependent translation [24–29] Yeast eIF4B is not essential, because knockout strains are viable but exhibit a temperature-sensitive and cold-sensitive phenotype [6,7] In addition, a new initiation factor termed eIF4H has been identified in mammals This Correspondence to R Rivera-Pomar, Centro Regional de Estudios ´ Genomicos, Av Calchaqui km 35, 500, 1888-Florencio Varela, Argentina Fax: + 54 (11) 4275 8379, Tel.: + 54 (11) 4275 8100, E-mail: rrivera@gwdg.de Abbreviations: eIF, eukaryotic initiation factor; PABP, poly(A)binding protein; IRES, internal ribosome entry site; RRM, RNA recognition motif; GST, glutathione S-transferase; RNAi, RNA interference (Received 11 October 2003, revised May 2004, accepted 14 May 2004) Keywords: cell survival; Drosophila; eukaryotic initiation factor 4B (eIF4B); proliferation; translation ´ 2924 G Hernandez et al (Eur J Biochem 271) factor shows sequence similarity to and functional conservation with human eIF4B [30] Schizosaccharomyces pombe SCE3 encodes an RNA-binding protein involved in cell division which has been found to share high sequence similarity with human eIF4B [31] In Drosophila, the mechanism of translational control is not well understood because of the lack of detailed information about the functional contribution of individual components of the translational machinery Only some of the factors involved in the initiation of translation have been characterized, including eIF4E [32,33], eIF4G [34,35], eIF4A [36] and eIF2 [37] The completion of the Drosophila genome project [38] has opened up the possibility of identifying most if not all translation factors Here we report the characterization of Drosophila melanogaster eIF4B gene We show that, in contrast with other analysed organisms, Drosophila possesses two eIF4B isoforms encoded by a single gene Both eIF4B isoforms are expressed throughout development, bind RNA, and stimulate capdependent synthesis of proteins in a redundant manner We also show that Dm-eIF4B is required for cell survival and that it stimulates cell proliferation Experimental procedures Construction of plasmids PCR amplification was performed on the EST LD09953 to introduce BamHI sites flanking the ORF of Dm-eIF4B-L The PCR product was cloned into pGEM-T (Promega) to create pGEMT-4BL, and into the BamHI site of the vectors pGEX-2T and pGEX-6P2 (Amersham Pharmacia Biotech) to create pGEX2T-4BL and pGEX6P2-4BL, respectively Derivatives corresponding to Dm-eIF4B-S were generated by mutagenesis on Dm-eIF4B-L plasmids using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) and the primers 5¢-GTGTCCAGGTGAATAACAGCTGGACG AGGAA-3¢ and 5¢-TTCCTCGTCCAGCTGTTATTCA CCTGGACAC-3¢ to introduce a stop codon in nucleotides 1171–1173 of the Dm-eIF4B-L ORF The yeast plasmids pTRP1-4BL, pTRP1-4BS and pHIS3-4A were generated by inserting Dm-eIF4B-L, Dm-eIF4B-S, and eIF4A [36] ORFs into the unique BamHI site of p301TRP1/GAL and p301-HIS3/GAL [7] The 5¢-UTR cDNA sequence of Ultrabithorax (Ubx) [39] and caudal [40] were cloned into the EcoRI site of pBluescript (Invitrogen) to create the constructs pBS-Ubx and pBS-Cad, respectively The 5¢-UTR cDNA sequence of Ubx was cloned into the SacI site of the pCap-FLuc (pLUC-cassette) vector which contains the Firefly luciferase (FLuc) ORF [41] to create the plasmid pUbx-FLuc The dicistronic reporter vector pFLuc/RLuc was generated by cloning the Renilla luciferase ORF (RLuc) into the HpaI site of the pLUCcassette The Ubx 5¢-UTR was also cloned into the BglII site of pFLuc/RLuc to create the construct pFLuc/Ubx/ RLuc Nucleotides 1215–1416 and 1311–1547 of the DmeIF4B-L and Dm-eIF4B-S cDNAs, respectively, were cloned into the EcoRV site of pBluescript vector to create the plasmids pBS-4BLduplex and pBS-4BSduplex The construct pUAS-4BL was generated by cloning the fulllength Dm-eIF4B-L cDNA from the EST LD09953 into the NotI–XhoI sites of pUAST [42] Ó FEBS 2004 Protein expression and purification, antibody production, and Western blot analysis In vitro transcription/translation of the ESTs was carried out using the TNT-coupled Reticulocyte Lysate System (Promega) in the presence of a [35S]Met and [35S]Cys mixture (14.3 mCiỈmL)1; Amersham) as described by the manufacturer Labeled proteins were resolved by SDS/ PAGE (12.5% gel) and detected by autoradiography Glutathione S-transferase (GST)-eIF4B-L and GST-eIF4B-S fusion proteins were expressed from pGEX6P2-4BL and pGEX6P2-4BS in Escherichia coli BL21 CodonPlus (Novagen) and purified with glutathione–Sepharose Fast Flow (Amersham Pharmacia Biotech) according to the manufacturers instructions The GST tag was removed by proteolytic digestion with PreScission Protease (Amersham Biosciences) and further purified on glutathione–Sepharose to remove both GST and protease GST-eIF4B-L was produced from pGEX2T-4B-L in E coli BL21(DE3)pLysS cells (Novagen) Polyclonal antibodies to Dm-eIF4B were generated in rabbit by immunization with 200 lg GSTeIF4B-L protein and Titer Max Adjuvant (Sigma) Lysates of wild-type Drosophila staged animals were freshly prepared by disrupting the samples on dry ice in a buffer containing 40 mM Hepes/KOH, pH 7.5, 200 mM KCl, mM EDTA, 1% (v/v) Triton X-100, 0.6 mL)1 aprotinin, 20 lgỈmL)1 leupeptin, 200 lgỈmL)1 soybean trypsin inhibitor, and EDTA-free protease inhibitor cocktail Complete (Roche Diagnostics GmbH), and centrifuged at 15 800 g for 10 at °C The supernatant was recovered on ice and immediately used without storing For Western blot analysis, lg total protein extracts were mixed with an equal volume of 2· sample buffer, boiled for min, and loaded on to a gel The protein concentration of the samples was quantified with the Protein-Assay Kit (Bio-Rad) Western blots were performed using rabbit anti(Dm-eIF4B) (1 : 20 000–1 : 80 000) and developed using the ECL detection kit (Amersham Pharmacia Biotech) Northern blot and quantitative real-time RT-PCR Total RNA of staged wild-type D melanogaster (Oregon R) was isolated using the RNeasy Mini Kit (Qiagen) and digested with RNase-free DNase I Northern blot was performed as described [33] The constructs pBS-4BLduplex and pBS-4BSduplex bearing isoform-specific sequences were linearized with PstI and transcribed with T3 RNA polymerase to synthezize 32P-labeled antisense RNAs Quantitative real-time RT-PCR was performed with 100 ng RNA and the QuantiTect SYBR green RT-PCR kit (Qiagen) in an Engine Opticon System (M.J Research Inc., Reno, NV, USA) Primers (25-mer) were designed to amplify 100 bp-long fragments Embryo whole-mount in situ hybridization and immunostaining Whole-mount in situ hybridization of embryos was performed as described [43] using antisense or sense (control) RNA probes against Dm-eIF4B-L (spanning nucleotides 1426–1215) or Dm-eIF4B-S (nucleotides 1547– 1311) mRNAs Images were acquired with an Axioplan Ó FEBS 2004 Functional analysis of Drosophila eIF4B (Eur J Biochem 271) 2925 Microscope coupled to a Kontron CCD camera Embryo immunohistochemistry was performed using rabbit anti(Dm-eIF4B) (1 : 500) and Cy3-labeled goat anti-rabbit Ig (Jackson, West Grove, PA, USA) Images were acquired with a CLS 310 confocal scanning microscope (Zeiss) In vivo complementation in Saccharomyces cerevisiae Constructs p301-TRP1/GAL1-TIF3 [16], pTRP1-4BL, pTRP1-4BS and pHIS3-4A were used to transform the yeast strain RCB-1C (MATa tif3::ADE2 ade2 his3 leu2 trp1 ura3 canR [6]) Cells were transformed using the lithium acetate method [44], plated and tested at 22 °C, 30 °C and 37 °C for growth complementation by Dm-eIF4B as described [45] RNA-binding assays Filter RNA-binding assays were performed using recombinant Dm-eIF4B isoforms as previously described [46] For cross-linking experiments, 32P-labeled RNA probes (Ubx and caudal 5¢-UTR) were generated by transcription of linearized pBS-Ubx and pBS-Cad with the T7 Mega transcription kit (Ambion, Austin, TX, USA) and radioactive ATP, GTP and UTP (Amersham) RNA probes were treated with RNase-free DNAse I (Qiagen), further purified using the RNeasy kit (Qiagen), and integrity was assessed by agarose gel electrophoresis RNA probes were diluted in 10 mM Hepes/K+, pH 7.6, containing 15 mM KCl and 2.5 mM MgCl2 The cross-linking was performed in 10 lL cross-linking buffer (10 mM Hepes/K+, pH 7.6, mM dithiothreitol, 5% glycerol, mM ATP, 100 ngỈlL)1 total yeast tRNA, and 10 lgỈlL)1 heparin), 1.6 lg Dm-eIF4B or GST protein and 100 000 c.p.m.ỈlL)1 RNA (previously treated for 15 at 70 °C) The reaction mixtures were incubated for 15 at room temperature and then irradiated for 35 on ice at 254 nM and digested for 45 with lL RNAse A (1 lgỈlL)1)/T1 (5 lL)1) at room temperature RNA–protein complexes were resolved in 10% SDS/PAGE and imaged in a Phosphoimager In vitro translation assays Translation extracts were prepared from 0–2 h-old Drosophila embryos as described [47] In vitro translation was performed as described [32,41] Translation assays in the presence of m7GpppG analog or from heat shockedembryos were as described [48] Transcripts were synthesized using the Ampliscribe mRNA transcription kit Reporter gene expression was determined using the Dualluciferase reporter assay system (Promega) and detected in a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA, USA) RNA interference (RNAi) Sense and antisense RNAs were prepared from linearized pBS-4BLduplex and pBS-4BSduplex using the Ampliscribe mRNA transcription kit (Biozym Diagnostics, Hessich Olendorf, Germany) in the presence of m7GpppG (New England BioLabs, Frankfurt am Main, Germany), digested with DNAse I and purified using the RNeasy kit Isoform- specific dsRNAs were produced by hybridization of an equimolar amount of sense and antisense RNAs in 50 mM NaCl and 20 mM Tris/HCl pH 8.0 (3 at 85 °C, 60 at 65 °C, chilled on ice and stored at )20 °C) The quality of the dsRNA molecules was monitored by agarose gel electrophoresis Drosophila Schneider S2 cells (1 · 106 in a 35-mm dish) were transfected with 7.5 lg dsRNA using the Effectene reagent (Qiagen) After 17 h, the medium was removed, the cells were resuspended in 4.5 mL medium, counted, and split into four dishes Then 24 h after transfection, the medium was removed from two wells and the cells subjected to starvation [medium containing 0.1% (v/v) fetal bovine serum] Two wells were kept in medium containing 10% (v/v) serum as fed controls At 48 h and 72 h after transfection (24 h and 48 h after starvation), the cells were harvested for counting and used for Western blotting using antibodies to Dm-eIF4B as described above Overexpression of Dm-eIF4B in S2 cells For the overexpression of Dm-eIF4B-L, · 106 Drosophila S2 cells in 35 mm dishes were transfected with either 100 ng pAct-Gal4 and 300 ng pUAS-4BL, or 100 ng pAct-Gal4 and 300 ng pUAS using the Effectene reagent (Qiagen) At 17 h after transfection, the medium was removed, and the cells were resuspended in 4.5 mL medium, counted, and split into four dishes At 24 h after transfection, the medium was removed from two wells, and the cells were subjected to starvation [medium containing 0.1% (v/v) fetal bovine serum] Two wells were kept in medium containing 10% (v/v) serum as fed controls At 48 h and 72 h after transfection (24 h and 48 h after starvation) the cells were counted Transgenic flies and overexpression analysis Flies were raised as described [49] The construct pUAS-4BL was used to generate transgenic flies as described [50] by microinjection in yw embryos Ectopic overexpession of eIF4B-L in imaginal eye discs was achieved using the Gal4 system [42] The transgenic strain yw; P{w UAS-eIF4B-L} (this study) was crossed to the strain w[*]; P{w[ + mC] ¼ GAL4-ninaE.GMR}12 (Bloomington Drosophila Stock Center), which drives expression of Gal4 in and behind the morphogenetic furrow of the developing eye imaginal disc [51] The F1 progeny was raised at 25 °C until thirdinstar larvae for imaginal disc analysis or until adulthood for phenotypic analysis Eye imaginal discs were dissected in · NaCl/Pi, fixed for 20 in 6% (v/v) formaldehyde/ · NaCl/Pi, blocked for h in 5% (v/v) normal horse serum in · NaCl/Pi, and immunostained at °C overnight with rabbit anti-(eIF4B-L) (1 : 7000) or rabbit anti(phospho-histone 3) (1 : 500; Upstate Biotechnology, Lake Placid, NY, USA) in 5% (v/v) normal horse serum/ · NaCl/Pi Discs were washed three times for 20 with · NaCl/Pi at room temperature and incubated for h with either Cy5-conjugated goat anti-rabbit Ig (1 : 1000) or Cy3-coupled donkey anti-rabbit Ig (1 : 250) Discs were further washed times for 20 in · NaCl/ Pi and, when indicated, incubated for with Alexa 488-conjugated phalloidin (1 : 100) in · NaCl/Pi Fluorescent signals were acquired and images analyzed using a ´ 2926 G Hernandez et al (Eur J Biochem 271) confocal scanning microscope LSM 510 Meta (Zeiss) and the apropriate filters set Adult flies were dehydrated in ethanol, dried, gold coated, and images acquired using a scanning electron microscope (Zeiss) as described [52] Results A single eIF4B gene encodes two eIF4B polypeptides in Drosophila A screening of Drosophila ESTs data base using the human eIF4B cDNA [5] revealed the existence of two groups of cDNAs, each with a distinct restriction pattern (data not shown) The comparison of these cDNAs with the genome of Drosophila [38] indicated the presence of a gene (CG10837) within the genomic contig AE003089 encoding the putative fly homolog of eIF4B D melanogaster eIF4B is a single-copy gene spanning 15.5 kb in the chromosome 3R within the Antp locus A detailed comparison of the sequences of each group of cDNAs with the genomic sequence supported the expression pattern of the eIF4B gene as proposed in Fig 1A The existence of one canonical polyadenylation signal for Dm-eIF4B-S pre-mRNA at nucleotide 9459, and three for Dm-eIF4B-L pre-mRNA at nucleotides 23144, 23148 and 23154 of the genomic fragment AE003089 gives rise to the synthesis of two pre-mRNAs of different length (Fig 1A) A subsequent alternative splicing (from nucleotide 9066 in the long premRNA) allows the specific removal of a premature termination codon (nucleotide 9071) from the long mRNA (Fig 1A) As a result of this expression pattern, we expected two mRNAs encoding two protein isoforms of Dm-eIF4B (termed Dm-eIF4B-L and Dm-eIF4B-S) Both mRNAs ( 1.5 and 1.6 kb long, respectively; not shown) have an identical short 5¢-UTR (39 nucleotides) but differ in their respective 3¢-UTRs (62 nucleotides for the long and 409 nucleotides for the short form) The ORFs predict a Dm-eIF4B-L isoform of 459 amino acids (52.2 kDa) and a Dm-eIF4B-S isoform of 390 amino acids (44.2 kDa) Interestingly, the two polypeptides share the first 389 amino acids, as the last amino acid of Dm-eIF4B-S is not present in Dm-eIF4B-L The alignment of the predicted amino-acid sequences of Dm-eIF4Bs with those of human [5] and yeast [6,7] counterparts shows a significant overall similarity, in particular in the N-terminal region (Fig 1B) A 50% similarity and 38% identity between the 346 first amino acids of Drosophila and human eIF4B is found Amino acids 75–277 of both Dm-eIF4Bs are 37% similar to and 27% identical with yeast eIF4B The similarity and identity between amino acids 90–346 of Dm-eIF4B and Sch pombe Sce3p are 44% and 36%, respectively, and 34% and 24% between amino acids 157–419 of Dm-eIF4B-L and both eIF4B1 from A thaliana and eIF4B from wheat, respectively (not shown) Amino acids 74–160 showed 29% identity with and 49% similarity to human eIF4H The RRM described for eIF4B homologs is conserved in Drosophila (Fig 1B, boxed), suggesting that both isoforms should be able to bind RNA On the other hand, the PABP-interacting motif [21] is not conserved, suggesting that the Dm-eIF4B and Dm-PABP not interact, as confirmed by our negative interaction results in vitro (not Ó FEBS 2004 shown) The region corresponding to the DRYG motif [5] is partially conserved A similar conservation pattern is observed in the putative Anopheles gambiae eIF4B gene ´ (G Hernandez and R Rivera-Pomar, unpublished observations) We analysed the size of the two Dm-eIF4B proteins by in vitro transcription/translation in rabbit reticulocyte lysate of cDNAs representative of each group of ESTs, which yielded two polypeptides with approximately the expected molecular mass (Fig 1C) To confirm the existence of two eIF4B protein isoforms in Drosophila cells, we raised antibodies against recombinant GST-eIF4B-L and performed Western blot experiments As shown in Fig 1D, the anti(Dm-eIF4B-L) serum was able to detect the recombinant proteins eIF4B-L (lane 4) and eIF4B-S (lane 5) as well as both endogenous Dm-eIF4B isoforms in S2 cell extracts (lane 6) The preimmune serum did not recognize the recombinant proteins or any polypeptide present in the cell extract (lanes 1–3) Developmental expression of the Dm-eIF4B gene The electrophoretic resolution of both mature eIF4B mRNA isoforms was not easily obtained when whole cDNA probes were used We observed a smear of 1.5– 1.6 kb, probably as a result of different poly(A) tails (Northern blot analysis; not shown) Thus, the occurrence and size of the transcripts was confirmed by Northen blotting using isoform-specific probes Developmental analysis revealed that both isoforms are expressed and detected as a single transcript from embryos to adulthood and that the isoform corresponding to eIF4B-S mRNA is the more abundant (Fig 2A) A significant maternal contribution was deduced from the high level of expression of eIF4B-S observed in very early embryos (0–3 h of development) The expression of Dm-eIF4B-L and DmeIF4B-S mRNAs was analyzed by quantitative real-time RT-PCR using total RNA derived from different life stages and specific oligonucleotide primers for each messenger (Fig 2B) Using this method, we observed a major contribution of eIF4B-S, which was on average four times more abundant than eIF4B-L We also performed Western blots using cell extracts from the same life cycle stages In agreement with our mRNA quantification experiments, we observed that both Dm-eIF4B-L and Dm-eIF4B-S proteins are expressed during the entire life cycle, but a higher level of Dm-eIF4B-S expression was detected (Fig 2C) In summary, a higher level of both Dm-eIF4B-S mRNA and protein with respect to Dm-eIF4B-L was detected in 0–3 h embryos Whole mount in situ hybridization in embryos using specific probes showed a ubiquitous but specific signal for Dm-eIF4B-L and Dm-eIF4B-S transcripts from early stages on (Fig 2D) A strong maternal component of both transcripts was observed (Fig 2D, syncytial blastoderm stage) At stages 14–16, they slightly accumulate in the nervous system (Fig 2D, stage 16) No signal was obtained when we used sense RNA probes as controls (Fig 2D, bottom) Immunohistochemistry and confocal imaging of embryos and cells in culture showed that the proteins are also ubiquitous (Fig 2E–J) However, as our antibody does not distinguish between isoforms, we cannot exclude the Ó FEBS 2004 Functional analysis of Drosophila eIF4B (Eur J Biochem 271) 2927 Fig A single gene encodes two eIF4B polypeptides in Drosophila (A) Gene structure of eIF4B The composition of the two pre-mRNAs was deduced from sequence comparison of the annotated gene (CG10837) (38) and ESTs LD09953 and LD14038, and confirmed by RT-PCR and sequencing of the exon–exon junctions Numbers refer to the genomic fragment AE003089 The first nucleotide of the donor splicing site of the second intron ( kb) in the long pre-mRNA is numbered (9066) Black boxes refer to the ORFs in each mRNA Stop codons are indicated by asterisks and numbered (9071 and 23122) The genes bcd and sd are located within the second intron and are transcribed in the opposite direction (B) Alignment of the deduced amino-acid sequences of Drosophila eIF4B-S and eIF4B-L with those of the human (h4B) [5] and yeast (y4B) [6,7] counterparts Conserved residues in all proteins or in two species are indicated as black and gray boxes, respectively Both elements of the RRM are squared (C) Autoradiography of [35S]Met incorporation in in vitro transcription/translation products of ESTs LD09953 (lane 1) and LD14038 (lane 2) Molecular mass markers are indicated on the left (D) Detection of both eIF4B isoforms in Drosophila cells Recombinant eIF4B-L (2 ng; lanes and 4), eIF4B-S (2 ng; lanes and 5) or S10 extracts (5 lg) from Schneider S2 cells (lanes and 6) were subjected to Western blot analysis using preimmune serum (lanes 1–3) or serum containing polyclonal antibodies against recombinant GST-eIF4B-L (lanes 4–6) (dilution : 20 000) Molecular mass protein markers are shown on the left ´ 2928 G Hernandez et al (Eur J Biochem 271) Ó FEBS 2004 Fig Expression of eIF4B during Drosophila life cycle and embryogenesis (A) Northern blot detection of Dm-eIF4B-S and Dm-eIF4B-L mRNAs during life cycle by isoform-specific probes (see Experimental procedures; also scheme in Fig 6A) Only a single mRNA of  1.6 kb was detected with each probe using total RNA derived from early embryos (0–3 h), embryos (0–18 h), first (1), second (2) and third (3) instar larvae, pupae (P) and adults (A) (B) Relative levels of Dm-eIF4B-S (dashed bars) and Dm-eIF4B-L (black bars) mRNAs measured by quantitative real-time RTPCR of total RNA from the stages shown in (A) The data represent three independent experiments (C) Detection of eIF4B-S and eIF4B-L by Western blot analysis of protein extracts prepared from the same developmental stages using anti-(GST-eIF4B-L) IgG (dilution : 20 000) Molecular mass markers are indicated on the right (D) Localization of Dm-eIF4B-L mRNA (left column) and Dm-eIF4B-S mRNA (middle column) during embryogenesis as revealed by in situ hybridization Developmental stages are indicated at the left side according to Campos-Ortega and Hartenstein (1997) [63] As a negative control, parallel in situ hybridization experiments were performed with sense probes and the same development time (lower panels) Embryos are oriented anterior to the left and dorsal to the top (E–J) Detection of Dm-eIF4B isoforms by immunohistochemistry of syncytial blastoderm (E), stage 11 embryo (F) and stage 14 embryo (G) Confocal sections corresponding to the embryos displayed in (E) and (F) show apical accumulation in the external and pole cells (H) and apical accumulation in tracheal pits (I) of the proteins (J) Immunohistochemistry of Schneider cells using anti-eIF4B IgG (dilution : 500) Ó FEBS 2004 Functional analysis of Drosophila eIF4B (Eur J Biochem 271) 2929 occurrence of isoform-specific distribution As expected, Dm-eIF4B is localized to the cytoplasm (Fig 2E–J) It is preferentially found in the apical region of polarized cells (Fig 2H–I) In cultured S2 cells, Dm-eIF4B-L and Dm-eIF4B-S are also strictly localized to the cytoplasm (Fig 2J) Transfection of S2 cells with C-terminal yellow fluorescent protein (YFP) fusions confirmed the cytoplasmic localization of both YFP-Dm-eIF4B-L and YFP-Dm-eIF4B-S (data not shown) Dm-eIF4B isoforms not substitute for yeast eIF4B in vivo We investigated whether both Dm-eIF4B isoforms alone or in combination with Drosophila eIF4A are able to complement the lack of eIF4B in a knockout yeast strain For this purpose, we cloned Dm-eIF4B-L, Dm-eIF4B-S and Dm-eIF4A ORFs into the yeast vectors p301-TRP1Gal1/10 and p301-HIS3-Gal1/10, which allow expression of cDNAs on galactose-containing media [16] as indicated in the Experimental procedures The constructs were introduced into the yeast strain RCB-1C, a temperaturesensitive and cold-sensitive null mutant of yeast eIF4B [6] In contrast with yeast eIF4B, none of the Dm-eIF4B isoforms alone or together with Dm-eIF4A were able to support a significant growth of the null mutant at 37 °C or 22 °C (data not shown) We also performed in vitro translation of a luciferase reporter mRNA using a cell-free translation yeast system derived from the eIF4B-deficient yeast strain [7] and supplemented it with our recombinant proteins In agreement with the in vivo results, the addition of Dm-eIF4B-L, Dm-eIF4B-S or Dm-eIF4A alone or in combinations showed no stimulation of luciferase synthesis (data not shown) Together, these results suggest that Dm-eIF4B is unable to replace the function of its yeast counterpart Binding of Dm-eIF4B-L and Dm-eIF4B-S to RNA Dm-eIF4B isoforms contain one putative RRM that is conserved in other eIF4Bs (Fig 1B, boxed) To test whether Dm-eIF4B binds to RNA, filter-binding assays [46] using purified Dm-eIF4B-L and Dm-eIF4B-S recombinant proteins and 32P-labeled RNA were performed Both isoforms bound to single-stranded RNA with similar affinity, with an estimated Kd,approx of 2.6 · 10)6 M and 2.8 · 10)6 M for Dm-eIF4B-L and Dm-eIF4B-S, respectively (Fig 3A) This value is significantly lower than the Kd,approx measured for Tif3p, which is in the range of 3.4 · 10)7 M in the same assays (not shown) Nevertheless, the lower values for Dm-eIF4B determined in our assays are consistent with the weak binding observed for human the eIF4B RRM domain [53] The RNA-binding activity of Dm-eIF4B was confirmed by cross-linking to the radiolabeled 5¢-UTR of Drosophila Ultrabithorax and caudal mRNA As shown in Figs 3B and 4C, both RNAs are cross-linked to Dm-eIF4B recombinant proteins, but not to GST Preliminary evidence for cross-linking of endogenous Dm-eIF4B to different RNAs was also ´ obtained using extracts from Drosophila S2 cells (S Lopez de Quinto, E Martı´ nez-Salas and J M Sierra, unpublished results) Fig Dm-eIF4B-L and Dm-eIF4B-S are RNA-binding proteins (A) Filter-binding assay showing the titration curves of Drosophila eIF4B proteins against 32P-labeled 38-nucleotide single-stranded RNA (0.25 pmol) [46] At the top, Kd,approx values are shown Seventy percent of the 38-nucleotide RNA is functional, as estimated by testing 1000 · molar excess of eIF4B over RNA For eIF4B-L and eIF4B-S, an approximate Kd ¼ 2.6 · 10)6 M and Kd ¼ 2.8 · 10)6 M, respectively, was estimated Owing to the extensive washing, the Kd values were rather underestimated (B, C) Cross-linking experiments were carried out in the absence of protein or the presence of GST, recombinant Dm-eIF4B-L or Dm-eIF4B-S proteins, with Ultrabithorax (B) or caudal (C) radiolabeled 5¢-UTRs (D) Coomassie staining of the gel shown in (C) Molecular mass markers are shown on the left Redundant function of Dm-eIF4B-L and Dm-eIF4B-S in translation To study the effect of Dm-eIF4B isoforms in cap-dependent and IRES-dependent translation, we analyzed the effect of both proteins on the translation of different mRNA reporters in a cell-free Drosophila embryo translation system [32,41] We used different in vitro transcribed, polyadenylated mRNA reporters, containing either the firefly (FLuc) or Renilla (RLuc) luciferase cistrons The cap-FLuc reporter [41] was used to monitor the cap-dependent translation as shown in Fig 4A Whereas the addition of BSA to the ´ 2930 G Hernandez et al (Eur J Biochem 271) Ó FEBS 2004 Fig Recombinant Dm-eIF4B-L and Dm-eIF4B-S enhance cap-dependent, but not IRES-dependent, translation in Drosophila cell-free extracts (A) Cap-dependent translation using cap-FLuc reporter mRNA in the absence or presence of increasing amounts of the indicated proteins (B) Functionality of the Ubx-IRES in a dicistronic reporter mRNA in vitro The effect of the addition of the cap analog m7GpppG on cap-dependent (black bars) and IRES-dependent (gray bars) translation using the dicistronic mRNAs (drawn on top) is shown (C) Effect of the addition of the indicated proteins on cap-dependent (black bars) and IRES-dependent (gray bars) translation using the dicistronic mRNA (drawn on top) shown FLuc, firefly luciferase; Ubx, Ultrabithorax IRES; RLuc, Renilla luciferase The data from four independent experiments are presented as percentage of control samples (no protein added) translation extracts did not have any effect on the translation of the reporter mRNA, translation of firefly luciferase (FLuc) mRNA increased up to twofold upon the addition of recombinant Dm-eIF4B-L, Dm-eIF4B-S upon an equimolecular mix of both to the translation system (Fig 4A), indicating a positive, specific and redundant effect on cap-dependent translation To assess the IRES-dependent translation, we performed similar experiments but using a capped dicistronic reporter mRNA bearing firefly luciferase (FLuc) as first cistron, and Renilla luciferase (RLuc) as the second cistron As an intercistronic element, we used the IRES of Ultrabithorax (Ubx), a well-defined IRES in Drosophila [39,54] To prove the functionality of the Ubx IRES in our system, we first performed competition experiments by addition of the cap analog m7GpppG to the translation reaction to mimic the lack of eIF4E in the lysate [48] Simultaneously we compared the translation of cistrons from both capped Ó FEBS 2004 Functional analysis of Drosophila eIF4B (Eur J Biochem 271) 2931 reporter FLuc/RLuc mRNA, lacking intercistronic sequences (negative control), and capped FLuc/Ubx/RLuc (Fig 4B) As expected, competition experiments with increasing cap analog concentrations and FLuc/RLuc (Fig 4B, left panel) showed that the efficiency of translation of the first cistron (black bars) decreased with increasing concentrations of cap analog, whereas the second cistron remained untranslated (gray bars) In the case of FLuc/ Ubx/RLuc mRNA (Fig 4B, right panel) translation of capped FLuc also decreased in the presence of the cap analog (black bars) Conversely, the presence of the cap analog enhanced the translation of the second cistron (RLuc, gray bars) to levels comparable to those obtained for the first cistron in the absence of cap inhibitor This indicated that the Ubx IRES is indeed driving internal initiation of the second cistron in our in vitro translational system We then analyzed the effect of the addition of recombinant eIF4B proteins or BSA as a control on FLuc/ Fig Inhibition of cap-dependent, but not IRES-dependent, translation by anti-eIF4B IgG and rescue of the translational activity by recombinant Dm-eIF4B proteins (A) Functionality of the Ubx IRES in the uncapped monocistronic mRNA reporter Ubx-FLuc Top, cap-FLuc and Ubx-FLuc mRNA reporters used Left, translation of cap-FLuc (circles) and Ubx-FLuc (squares) in the presence of cap analog m7GpppG Right, translation of cap-FLuc (black bars) and Ubx-FLuc (gray bars) in extracts prepared from heat-shock-treated embryos (B–D) Differential inhibition of cap-dependent vs IRES-dependent translation after incubating extracts with antibodies against Drosophila eIF4B Translation of the monocistronic reporter mRNAs cap-FLuc (B), Ubx-FLuc (C) and the dicistronic capped FLuc/Ubx/ RLuc (D) in the absence or presence of purified IgG fraction from preimmune serum or from serum containing anti-eIF4B IgG In (D) cap-dependent and IRES-dependent translation values are indicated as black and gray bars, respectively (E) Rescue of the translational activity of an eIF4B-immunodepleted extract by addition of recombinant proteins Dm-eIF4B-L and Dm-eIF4B-S The data from four independent experiments are represented as percentage of the control samples without the addition of protein Ubx/RLuc mRNA in our translational assay Simultaneous determination of both cap-dependent and IRES-dependent initiation indicated preferential translation of the first cistron when eIF4B-L or eIF4B-S, but not BSA was added (Fig 4C, black bars) However, no significant effect on IRES-dependent initiation was detected upon addition of the Dm-eIF4B proteins or BSA (Fig 4C, gray bars) Similar results were obtained using the IRES of Antennapedia [55] (data not shown) We conclude that cap-dependent initiation is enhanced by Dm-eIF4B-L and Dm-eIF4B-S and that their effect on cap-dependent translation is equivalent Our results also indicate that IRES-dependent translation is not affected by either form of Dm-eIFB We also wanted to prove the functionality of Ubx IRES when using a monocistronic reporter mRNA Ubx-FLuc (see below) For this purpose, we performed cap analog competition experiments and compared the translational efficiency of noncapped Ubx-FLuc reporter mRNA with ´ 2932 G Hernandez et al (Eur J Biochem 271) that of cap-FLuc (Fig 5A, left panel) Addition of 0.1 mM cap analog resulted in a 60% inhibition of translation of cap-FLuc (circles), whereas that of uncapped Ubx-FLuc remained unchanged (squares) Further addition of cap analog resulted in 90% inhibition of cap-FLuc mRNA translation Conversely, translation of Ubx-FLuc increased in the presence of cap analog, reaching a plateau at 140% of translation In parallel, we tested these reporters in translation extracts derived from untreated and heat-shocked embryos [48] (Fig 5A, right), e.g under conditions when cap-dependent initiation is severely impaired [48,56] In extracts derived from untreated embryos, both cap-FLuc and Ubx-FLuc mRNAs were efficiently translated (control) In translation extracts derived from heat-shocked embryos (heat-shocked) translation of cap-FLuc decreased to 30% of the control (untreated extracts; black bars), whereas that of Ubx-FLuc mRNA increased five times (gray bars) These results confirm the ability of Ubx-FLuc mRNA to be translated in an IRES-dependent manner and were used in further experiments We translated cap-FLuc and Ó FEBS 2004 Ubx-FLuc mRNA in the presence of purified IgG against Dm-eIF4B-L (Fig 5B–D) Addition of anti-(Dm-eIF4B-L) IgG (but not IgG purified from preimmune serum) resulted in a twofold decrease in cap-FLuc translation (Fig 5B) Conversely, anti-(Dm-eIF4B-L) IgG had only a minor effect on translation of Ubx-FLuc mRNA (Fig 5C) Translation of the first cistron of the dicistronic transcript FLuc/Ubx/RLuc (Fig 5D) was also affected by anti-(DmeIF4B-L), but had no significant effect on IRES-dependent translation of the second cistron (Fig 5D) Inhibition of cap-dependent translation caused by the anti-(Dm-eIF4BL) IgG could be reversed to some extent by the addition of recombinant Dm-eIF4B-L or Dm-eIF4B-S to the cell-free extract (Fig 5E) The relative stimulation of luciferase synthesis obtained in the Dm-eIF4B-inhibited lysate was similar to that obtained in the noninhibited lysate Taken together, these results suggest that both Dm-eIF4B isoforms have a redundant positive effect on cap-dependent mRNA translation but not intervene in IRES-dependent translation Fig Drosophila eIF4B is required for cell survival (A) Localization of the dsRNAs (arrows) used to knock down each eIF4B transcript (B) Levels of eIF4B isoforms in Schneider S2 mock cells (lane C) or in cells transfected with dsRNA specific for Dm-eIF4B-L (lanes L and L + S) or Dm-eIF4B-S (lanes S and L + S) Total protein (1 lg per lane) from cells incubated for 48 h after transfection was probed with anti-(Dm-eIF4B-L) IgG (C) Effect of RNAi on growth on fed (10% fetal bovine serum) S2 cells The cells were mock transfected (circles) or transfected with a mixture of the dsRNAs for both Dm-eIF4B isoforms (squares) and, after 24 h, re-plated in the same medium containing 10% fetal bovine serum and incubated for 24 h or an additional 48 h The number of viable cells at the time of re-plating (0 h) is taken as 100% (D) Protein synthesis in fed (10% fetal bovine serum) eIF4B knockdown S2 cells Aliquots of the cells incubated for 48 h (C) were pulsed with [35S]methionine (50 lCiỈmL)1) for h, and the labeled proteins analysed by SDS/PAGE (lower panel) Numbers correspond to the incorporation of [35S]methionine The levels of eIF4B isoforms were estimated by Western blot (upper panel) (E) Effect of RNAi on growth of starved (0.1% fetal bovine serum) S2 cells The experiment was carried out as in (C) except that the cells were re-plated in medium containing 0.1% fetal bovine serum All experiments were performed in triplicate Ó FEBS 2004 Functional analysis of Drosophila eIF4B (Eur J Biochem 271) 2933 Dm-eIF4B is involved in cell survival and stimulates cell proliferation To determine the in vivo requirement for Dm-eIF4B, we used RNAi to knock-down Dm-eIF4B in Drosophila S2 cells [57] Specific dsRNAs for each Dm-eIF4B mRNA isoform were used in our transfection experiments (for a scheme, see Fig 6A) An efficient reduction of Dm-eIF4B expression was observed by Western blot analysis Dm-eIF4B-L (Fig 6B, lane L) and most of Dm-eIF4B-S, the more abundant isoform (Fig 6B, lane S), were knocked down 48 h after transfection with specific dsRNAs Surprisingly, the Dm-eIF4B-S dsRNA also resulted in a reduction in the levels of the Dm-eIF4B-L (Fig 6B, lane S) Whether this was due to a direct effect of the interfering RNA or a secondary effect on the synthesis of eIF4B-L by the removal of eIF4B-S could not be established However, the simultaneous transfection with both dsRNAs was effective in completely removing Dm-eIF4B-L and 90% of Dm-eIF4B-S at 48 h (Fig 6B, lane L + S) and 72 h after transfection Doubletransfected cells were used for a parallel analysis of cell growth and protein synthesis Removal of both eIF4B isoforms resulted in a slight inhibitory effect on cell proliferation in the presence of 10% serum (Fig 6C) This is analogous to the knockout phenotype observed in yeast, where lack of Tif3 is not essential for cell viability and has only a moderate effect on cell proliferation at 30 °C Accordingly, a small but reproducible effect on protein synthesis (measured as incorporation of methionine) was observed (Fig 6D) Interestingly, when knocked-down cells were grown at low serum concentrations (0.1%), a significant reduction in cell survival was observed (Fig 6E) From our results we conclude that, under optimal growth conditions (10% serum), eIF4B is not essential, but it significantly stimulates cell proliferation under limiting growth conditions (0.1% serum) A more drastic effect was observed by overexpressing Dm-eIF4B-L S2 cells were cotransfected with plasmid pUAS-4B-L, a vector bearing the cDNA of Dm-eIF4B-L under the control of a UAS promoter, and plasmid pActinGAL4 to activate expression of Dm-eIF4B-L (Fig 7A) Transfected cells stimulated proliferation independently of the serum concentration used (Fig 7B) We also investigated if the overexpression of Dm-eIF4B-L was able to alter the pattern of cell proliferation in eye imaginal discs The Drosophila eye develops from the imaginal discs as a very precisely patterned structure, which is a powerful tool for studying cell proliferation and differentiation [58] In the developing eye imaginal disc, there are two waves of cells undergoing mitosis with a dynamic structure between them, the morphogenetic furrow The morphogenetic furrow separates a region of differentiated cells from a region of proliferating cells [58] (Fig 7C, arrow) We produced transgenic flies bearing the same construct as the one previously used in overexpression experiments in S2 cells and genetically manipulated transgenic descendants to express Dm-eIF4B-L in the differentiating part of the eye imaginal disc (for details see Experimental procedures) Immunofluorescence and Western blot experiments confirmed the overexpression of Dm-eIF4B-L in the differentiated, nonproliferative tissue (Fig 7C, left and center panels) We estimated that the level of Dm-eIF4B-L Fig Effect of overexpression of Dm-eIF4B-L in S2 cells and imaginal discs (A) Plasmids used in this study Act, constitutive Actin5C promoter; GMR, glass promoter, which drives gene expression in the posterior region of the morphogenetic furrow of the developing eye imaginal disc; UAS, upstream activator sequence; 4BL, Dm-eIF4B-L cDNA (B) Effect of overexpression of Dm-eIF4B-L on cell proliferation of fed (10% serum) or starved (0.1% serum) S2 cells The experiment was carried out as in Fig except that cells were cotransfected with pAct-Gal4 and either pUAS-4BL or pUAS (control) (C) Overexpression of eIF4B-L in eye imaginal discs Immunodetection of eIF4B in wild-type (left) or transgenic UAS-4BL/GMR (middle) eye antennal imaginal discs Arrows indicate the morphogenetic furrow of the eye imaginal disc The level of eIF4B-L was estimated by Western blot in wild-type (1; 20 discs) or UAS-4BL/GMR transgenic (2; 15 discs) eye imaginal discs (D) Scanning electron microscopy (200 ·) of an adult eye from wild-type (left) or UAS-4BL/GMR transgenic (right) flies Note the disarray of the otherwise patterned structure of the eye, the increased size of the ommatidia, and the presence of extra chaeta (E) As in (D) but at 2000 · magnification (F) Staining of mitotic cells in wildtype (left) or UAS-4BL/GMR transgenic (middle) eye antennal imaginal discs with anti-(phosphorylated histone 3) Ig (red) Actin was labeled with Alexa-488-conjugated phalloidin (green) Extramitotic cells in the differentiated tissue, posterior to the morphogenetic furrow (arrows), are evident in the disc overexpressing eIF4B-L Asterisks indicate the two waves of mitotic cells Discs are oriented anterior to the left ´ 2934 G Hernandez et al (Eur J Biochem 271) expression increased 10 times relative to the expression of eIF4B-S (Fig 7C, right panel) The analysis of the adult eye showed abnormal growth and disruption of the periodic pattern of ommatidia (Fig 7D) with a penetrance > 70% This is consistent with overproliferation phenotypes, a fact reinforced by the presence of extra chaete (Fig 7E) In the wild-type eye, there is a periodic pattern of individual chaete surrounding one ommatidia (Fig 7E, left panel) In the transgenic eye, we consistently observed the presence of groups of two to three chaeta (Fig 7E, right panel) As these structures derive from a single cell in the differentiation region, we conjectured that the observed phenotype may be the result of extra mitosis rounds in an otherwise nonproliferative tissue To confirm this hypothesis, we analysed the pattern of cell proliferation in the developing eye by in situ detection of phosphorylated histone 3, which specifically marks mitotic cells and filamentous actin allowing the regular pattern of the differentiating tissue to be seen (a scheme of the eye imaginal disc is shown in the right panel; the arrow indicates the morphogenetic furrow and the two mitosis waves) In the wild-type eye imaginal disc, cell division is restricted to the antennal disk and to the two mitotic waves flanking the morphogenetic furrow (Fig 7F, left panel) The discs overexpressing Dm-eIF4B-L display additional rounds of mitosis, in particular in the otherwise nondividing cells posterior to the morphogenetic furrow (Fig 7F, middle panel) The rate of proliferation observed in cells expressing higher levels of Dm-eIF4B-L precisely correlated with the eye phenotype Discussion We have characterized two isoforms of eIF4B from D melanogaster, Dm-eIF4B-L and Dm-eIF4B-S, which are encoded by a single gene and generated by the use of two alternative polyadenylation sites and subsequent alternative splicing of the second intron This likely reflects similarities to human eIF4B, which is encoded by two different mRNAs also produced by alternative polyadenylation sites, although both mRNAs encode the same polypeptide [5] In S cerevisiae [6,7] and wheat [9], a single eIF4B polypeptide is produced, whereas in A thaliana two eIF4B isoforms are encoded by two different genes [9] In Anopheles gambiae, only a single gene could be detected from the genome ´ sequence (G Hernandez and R Rivera-Pomar, unpublished observation) The presence of most initiation factors in all eukaryotic organisms and the high sequence similarity across the phyla are evidence of the evolutionary conservation of the translational machinery Human and Drosophila eIF4E are able to substitute for their yeast homolog in vivo ([45]; ´ G Hernandez, M Altmann and R Rivera-Pomar, unpublished data) However, not all factors exhibit this property; mammalian and Drosophila eIF4A and Drosophila eIF4G are not able to substitute for their yeast counterparts ([59]; ´ G Hernandez, M Altmann and R Rivera-Pomar, unpublished data) This is also true for Dm-eIF4B It may reflect the existence of subtle differences in the basic translational machinery between species, both concerning the required components and their interactions Another explanation could be that the lower affinity for RNA of Dm-eIF4B, which also accounts for the mammal counterpart, is not Ó FEBS 2004 sufficient to fulfil its function in yeast Furthermore, factors involved in more complex interactions with other proteins may require the presence of species-specific components, a subject that deserves future investigation In addition to its role in cap-dependent translation, human eIF4B is involved in IRES-dependent translation of picornaviral mRNAs [24,26–29] In this study we observed that both Dm-eIF4B isoforms preferentially promote capdependent translation, which is sensitive to immunodepletion and the addition of recombinant protein Dm-eIF4B As the excess of eIF4B or the presence of anti-(Dm-eIF4B) IgG did not show a significant effect on the in vitro activity of Ultrabithorax and Antennapedia IRES, we conclude that, at least for these mRNAs, eIF4B might not be involved In vitro binding of Dm-eIF4B to these two IRESs is likely due to unspecific RNA-binding activity Whether this implies a more important role of eIF4B for the translation of viral rather than for cellular IRESs or, alternatively, other Drosophila IRESs are eIF4B-dependent has not been established and requires further investigation However, we can clearly conclude that eIF4B has a role in capdependent translation in Drosophila embryonic extracts It has been proposed that eIF4B, eIF4A and eIF4F are involved in the scanning process complementing the role of eIF1 and eIF1A, and that in the presence of eIF4F, eIF4B and eIF4A, scanning is less dependent on eIF1 and eIF1A [60,61] We observed that reduction of eIF4B decreases the level of cap-dependent translation while addition of recombinant eIF4B increases translation activity in vitro, arguing for a central although not exclusive role of eIF4B in the initiation process Our depletion experiments support this conclusion Deletion of yeast Tif3 has been shown to cause slow growth and temperature sensitivity in yeast cells [6,7] Our RNAi-based knock-down experiments in cultured cells show reduced effects on the rate of protein synthesis and, accordingly on cell proliferation This suggests that in higher eukaryotes also (as shown for yeast) eIF4B is not essential, although we cannot exclude the possibility that the residual level of Dm-eIF4B after RNAi is enough to keep Drosophila S2 cells alive Under starvation conditions, i.e a situation in which the cells not divide, levels of endogenous Dm-eIF4B were reduced four times (data not shown) As RNAi treatment performed in S2 cells during starvation showed that eIF4B is required for cell survival, additional eIF4B reduction by RNAi treatment may explain why the cells died In yeast, eIF4A is required for cell survival during starvation [62], suggesting that its helicase activity is required to survive longer periods of treatment, as observed here for Dm-eIF4B Overexpression of Dm-eIF4B-L in Drosophila cell cultures and in developing tissues provided interesting insights into the function of eIF4B In mammalian cell cultures, a 50-fold overexpression of eIF4B resulted in the inhibition of the protein synthesis of a reporter mRNA [5] and no obvious phenotype Here we show that a 10-fold increase in Dm-eIF4B-L promotes cell proliferation in differentiated imaginal tissues Although we could not prove that this is a direct effect due to enhanced translation rates, it correlates with our in vitro experiments demonstrating enhanced translation by the addition of recombinant Dm-eIF4B-L We provide here the first in vivo evidence for increased cell proliferation in response to changes in eIF4B Ó FEBS 2004 Functional analysis of Drosophila eIF4B (Eur J Biochem 271) 2935 levels This opens up the way for further biochemical and genetics experiments to elucidate the mode of action of eIF4B 13 Acknowledgements ´ We thank Annelies Zechel for excellent technical assistance, Jose Alcalde for antibody production, Marian Bienz for the Ubx cDNA, ´ Fatima Gebauer for the pLUC-cassette vector, Iris Plischke for the injection of embryos, Peter Schwartz for the scanning electronic microscopy of the samples, Martin Zeidler and Tina Mukherjee for sharing their knowledge on eye development, and Stephan Hoppner for ă proofreading the manuscript This work was supported by the Max Planck Gesellschaft and the Bundesministerium fuer Bildung und ´ Forschung (R.R.-P.), and by grants from the Ministerio de Educacion y ´ ´ Cultura and Fundacion Ramon Areces to the CBM, Spain (J.M.S.) 14 15 16 References Hershey, J.W.B & Merrick, W.C (2000) The pathway and mechanism of inititiation of protein synthesis In Translational Control of Gene Expression (Sonenberg, N., Hershey, J.W.B & Mathews, M.B., eds), pp 33–88 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Gingras, A.C., Raught, B & Sonenberg, N (1999) eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation Annu Rev Biochem 68, 913–963 Jackson, R.J (2000) Comparative view of initiation site selection mechanisms In Translational Control of Gene Expression (Sonenberg, N., Hershey, J.W.B & Mathews, M.B., eds), pp 127– 184 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 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Coomassie staining of the gel shown in (C) Molecular mass markers are shown on the left Redundant function of Dm-eIF4B-L and Dm-eIF4B-S in translation To study the effect of Dm-eIF4B isoforms in cap-dependent. .. cultures and in developing tissues provided interesting insights into the function of eIF4B In mammalian cell cultures, a 50-fold overexpression of eIF4B resulted in the inhibition of the protein synthesis... whether Dm-eIF4B binds to RNA, filter-binding assays [46] using purified Dm-eIF4B-L and Dm-eIF4B-S recombinant proteins and 32P-labeled RNA were performed Both isoforms bound to single-stranded RNA