eIF4B-L and eIF4B-S preferentially enhance cap-dependent over IRES-dependent translation initiation in a Drosophila cell-free translation system.. To confirm the existence of two eIF4B pr
Trang 1Two functionally redundant isoforms of Drosophila melanogaster
eukaryotic initiation factor 4B are involved in cap-dependent
translation, cell survival, and proliferation
Greco Herna´ndez1, Paula Va´zquez-Pianzola1, Andreas Zurbriggen2, Michael Altmann2, Jose´ M Sierra3 and Rolando Rivera-Pomar1
1
Max-Planck-Institute fu¨r biophysikalische Chemie, Go¨ttingen, Germany;2Institut fu¨r Biochemie und Molekularbiologie,
Universita¨t 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
tran-scription 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 cell-free 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
Keywords: cell survival; Drosophila; eukaryotic initiation factor 4B (eIF4B); proliferation; translation
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 ATP-dependent 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 inter-action 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
Geno´micos, 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 1 May 2004,
accepted 14 May 2004)
Trang 2factor shows sequence similarity to and functional
conser-vation 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
eIF4Bgene We show that, in contrast with other analysed
organisms, Drosophila possesses two eIF4B isoforms
enco-ded by a single gene Both eIF4B isoforms are expressed
throughout development, bind RNA, and stimulate
cap-dependent 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
Quik-Change Site-Directed Mutagenesis Kit (Stratagene) and the
primers 5¢-GTGTCCAGGTGAATAACAGCTGGACG
AGGAA-3¢ and 5¢-TTCCTCGTCCAGCTGTTATTCA
CCTGGACAC-3¢ to introduce a stop codon in
nucleo-tides 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
p301-TRP1/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
lucif-erase ORF (RLuc) into the HpaI site of the
pLUC-cassette 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
Dm-eIF4B-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
full-length Dm-eIF4B-L cDNA from the EST LD09953 into
the NotI–XhoI sites of pUAST [42]
Protein expression and purification, antibody production, and Western blot analysis
In vitrotranscription/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 mCiÆmL)1; Amersham) as described by the manufacturer Labeled proteins were resolved by SDS/ PAGE (12.5% gel) and detected by autoradiography Gluta-thione 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 4 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 GST-eIF4B-L protein and Titer Max Adjuvant (Sigma) Lysates
of wild-type Drosophila staged animals were freshly pre-pared by disrupting the samples on dry ice in a buffer containing 40 mM Hepes/KOH, pH 7.5, 200 mM KCl,
4 mMEDTA, 1% (v/v) Triton X-100, 0.6 UÆmL)1 aproti-nin, 20 lgÆmL)1 leupeptin, 200 lgÆmL)1 soybean trypsin inhibitor, and EDTA-free protease inhibitor cocktail Com-plete (Roche Diagnostics GmbH), and centrifuged at
15 800 g for 10 min at 4C The supernatant was recovered
on ice and immediately used without storing For Western blot analysis, 5 lg total protein extracts were mixed with
an equal volume of 2· sample buffer, boiled for 2 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-mountin 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
Trang 3Microscope 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 22C, 30 C and
37C for growth complementation by Dm-eIF4B as
described [45]
RNA-binding assays
Filter RNA-binding assays were performed using
recom-binant 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
radio-active 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 mMMgCl2 The cross-linking was performed in 10 lL
cross-linking buffer (10 mM Hepes/K+, pH 7.6, 1 mM
dithiothreitol, 5% glycerol, 1 mM ATP, 100 ngÆlL)1total
yeast tRNA, and 10 lgÆlL)1 heparin), 1.6 lg Dm-eIF4B
or GST protein and 100 000 c.p.m.ÆlL)1RNA (previously
treated for 15 min at 70C) The reaction mixtures were
incubated for 15 min at room temperature and then
irradiated for 35 min on ice at 254 nM and digested for
45 min with 1 lL RNAse A (1 lgÆlL)1)/T1 (5 UÆlL)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
Droso-phila embryos as described [47] In vitro translation was
performed as described [32,41] Translation assays in the
presence of m7GpppG analog or from heat
shocked-embryos were as described [48] Transcripts were
synthes-ized using the Ampliscribe mRNA transcription kit
Reporter gene expression was determined using the
Dual-luciferase 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 mMTris/HCl pH 8.0 (3 min at 85C, 60 min
at 65C, 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· 106in 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 transfec-tion, 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, 1· 106Drosophila 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 25C until third-instar larvae for imaginal disc analysis or until adulthood for phenotypic analysis Eye imaginal discs were dissected
in 1· NaCl/Pi, fixed for 20 min in 6% (v/v) formaldehyde/
1· NaCl/Pi, blocked for 1 h in 5% (v/v) normal horse serum in 1· NaCl/Pi, and immunostained at 4C over-night with rabbit (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/
1· NaCl/Pi Discs were washed three times for 20 min with 1· NaCl/Piat room temperature and incubated for
2 h with either Cy5-conjugated goat anti-rabbit Ig (1 : 1000) or Cy3-coupled donkey anti-rabbit Ig (1 : 250) Discs were further washed 3 times for 20 min in 1· NaCl/
Pi and, when indicated, incubated for 8 min with Alexa 488-conjugated phalloidin (1 : 100) in 1· NaCl/Pi Fluor-escent signals were acquired and images analyzed using a
Trang 4confocal 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 singleeIF4B gene encodes two eIF4B polypeptides
inDrosophila
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
pre-mRNA) 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,
respect-ively (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 do not interact, as
confirmed by our negative interaction results in vitro (not
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 Herna´ndez and R Rivera-Pomar, unpublished obser-vations)
We analysed the size of the two Dm-eIF4B proteins by
in vitrotranscription/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 anti-bodies 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 theDm-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 expres-sion of eIF4B-S observed in very early embryos (0–3 h of development) The expression of eIF4B-L and Dm-eIF4B-S mRNAs was analyzed by quantitative real-time RT-PCR using total RNA derived from different life sta-ges and specific oligonucleotide primers for each messen-ger (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 West-ern 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
Trang 5Fig 1 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 ( 9 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 1 and 4), eIF4B-S (2 ng; lanes 2 and 5) or S10 extracts (5 lg) from Schneider S2 cells (lanes 3 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 1 : 20 000) Molecular mass protein markers are shown on the left.
Trang 6Fig 2 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 RT-PCR 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 1 : 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 1 : 500).
Trang 7occurrence 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
cyto-plasmic localization of both YFP-Dm-eIF4B-L and
YFP-Dm-eIF4B-S (data not shown)
Dm-eIF4B isoforms do 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-TRP1-Gal1/10 and p301-HIS3-p301-TRP1-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
temperature-sensitive and cold-temperature-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 37C
or 22C (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
recom-binant 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)6M and
2.8· 10)6M for Dm-eIF4B-L and Dm-eIF4B-S,
respect-ively (Fig 3A) This value is significantly lower than the
Kd,approx measured for Tif3p, which is in the range of
3.4· 10)7M in the same assays (not shown)
Neverthe-less, 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
caudalmRNA 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 Lo´pez
de Quinto, E Martı´nez-Salas and J M Sierra,
unpub-lished results)
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, polyadenyl-ated mRNA reporters, containing either the firefly (FLuc)
or Renilla (RLuc) luciferase cistrons The cap-FLuc repor-ter [41] was used to monitor the cap-dependent translation
as shown in Fig 4A Whereas the addition of BSA to the
Fig 3 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, K d,approx values are shown Seventy per-cent 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 K d ¼ 2.6 · 10)6M and K d ¼ 2.8 · 10)6M , respect-ively, was estimated Owing to the extensive washing, the K d values were rather underestimated (B, C) Cross-linking experiments were carried out in the absence of protein or the presence of GST, recom-binant 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.
Trang 8translation extracts did not have any effect on the
transla-tion of the reporter mRNA, translatransla-tion of firefly luciferase
(FLuc) mRNA increased up to twofold upon the addition
of recombinant Dm-eIF4B-L, Dm-eIF4B-S upon an
equi-molecular 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
Fig 4 Recombinant Dm-eIF4B-L and Dm-eIF4B-S enhance cap-dependent, but not IRES-dependent, translation in Drosophila cell-free extracts (A) Cap-dependent transla-tion 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 m 7 GpppG on cap-dependent (black bars) and IRES-dependent (gray bars) trans-lation 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) trans-lation 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).
Trang 9reporter 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/
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 initi-ation 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
Fig 5 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)
Func-tionality 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)
Dif-ferential 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
puri-fied IgG fraction from preimmune serum or
from serum containing anti-eIF4B IgG In (D)
cap-dependent and IRES-dependent
transla-tion values are indicated as black and gray
bars, respectively (E) Rescue of the
transla-tional 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
rep-resented as percentage of the control samples
without the addition of protein.
Trang 10that 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
trans-lation 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
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-(Dm-eIF4B-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-eIF4B-L) 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 do not intervene in IRES-dependent translation
Fig 6 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 incu-bated 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 knock-down S2 cells Aliquots of the cells incubated for 48 h (C) were pulsed with [35S]methionine (50 lCiÆmL)1) for 2 h, and the labeled pro-teins analysed by SDS/PAGE (lower panel) Numbers correspond to the incorporation of [ 35 S]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 con-taining 0.1% fetal bovine serum All experi-ments were performed in triplicate.