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Molecular & Biochemical Parasitology 155 (2007) 146–155

Characterization of human malaria parasite Plasmodium falciparumeIF4E homologue and mRNA 5′ cap status

Philip J. Shaw ∗, Napawan Ponmee, Nitsara Karoonuthaisiri,Sumalee Kamchonwongpaisan, Yongyuth Yuthavong

National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency,113 Pahonyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand

Received 27 November 2006; received in revised form 1 June 2007; accepted 5 July 2007Available online 10 July 2007

bstract

The mRNA 5′ cap is an essential structural feature for translation of eukaryotic mRNA. Translation is initiated by recognition of the cap by theranslation initiation factor eIF4E. To further our understanding of mRNA translation in the human malaria parasite Plasmodium falciparum, weave investigated the parasite eIF4E and its interaction with capped mRNA. We have purified P. falciparum eIF4E as a recombinant protein andemonstrated that it has canonical mRNA cap binding activity. We used this protein to purify P. falciparum capped mRNAs from total parasite

NA. Microarray analysis comparing total and eIF4E-purified capped mRNAs shows that 34 features were more than twofold under-represented

n the purified RNA sample, including 19 features representative of nuclear transcripts. The putatively uncapped nuclear transcripts may representclass of mRNAs targeted for storage and cap removal.2007 Elsevier B.V. All rights reserved.

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eywords: Malaria; eIF4E; 5′ cap; Microarray; mRNA

. Introduction

Translation initiation is a complicated process involvingany proteins and different mRNA structural features [1]. The

′ end of nascent eukaryotic mRNA transcribed by RNA poly-erase II in the nucleus is modified to have a m7G(5′)ppp(5′)N

ap structure, which has important roles in RNA splicing, sta-ility, nuclear export and translation initiation [2]. The cap isecognized by the eIF4E protein, a part of the eIF4F cap bind-ng protein complex. eIF4E’s specificity for the cap structurever other nucleotides has been studied extensively; for exam-le, structural studies of eIF4E bound to 7-methyl-GDP [3]ave shown how the cap-characteristic N7-methyl group makesritical van der Waals contacts with the eIF4E Trp166 residueconserved across species [4]).

eIF4E homologues have been characterized in a wide rangef eukaryotic species, including parasitic protists such as Leish-ania major [5,6] and Giardia lamblia [7]. Some of these eIF4E

∗ Corresponding author. Tel.: +66 2 564 6700; fax: +66 2 564 6707.E-mail address: philip@biotec.or.th (P.J. Shaw).

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166-6851/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.molbiopara.2007.07.003

omologues lack cap binding activity and their roles are unclear.ost-transcriptional regulatory mechanisms can interfere with

he eIF4F complex assembly, either through eIF4E binding pro-eins [8], or by specific mRNA decay in which the mRNA ise-capped [9].

The human malaria parasite Plasmodium falciparum capsts mRNAs with a canonical cap structure, as it has functional

RNA capping enzymes [10] and cDNA cloning of full-lengthDNAs by “oligo capping” has been demonstrated, indicatinghe presence of capped mRNA [11]. In contrast, few details arenown of P. falciparum translation, including functional char-cterization of its translation factors. By comparison with otherukaryotes, we hypothesized that P. falciparum has an eIF4Eith canonical cap binding activity. In this paper, we report the

loning and expression of P. falciparum eIF4E as a GST-taggedrotein. Cap binding of this protein was demonstrated by initro assays and parasite capped RNA could be purified withhe protein, thereby confirming our hypothesis. P. falciparum

icroarrays were employed to compare transcript abundanceetween total and purified capped RNA samples, and a subsetf mRNAs under-represented in the purified sample was demon-trated. This subset contains RNAs derived from the plastid and

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itochondrial genomes, and also unexpectedly, from nuclearenes. The under-represented nuclear mRNAs may constituteclass of predominantly uncapped mRNAs that are perhaps

electively targeted for storage and cap removal.

. Materials and methods

.1. Cloning and expression of the P. falciparum eIF4Eomologue

The P. falciparum gene PFC0635c has previously beenescribed as a putative eIF4E homologue [4]. BLAST searchsing the encoded protein sequence, or eIF4E homologues fromther species, did not reveal additional homologues. The codingequence of PFC0635c was cloned by PCR using primers′-AAAAGGATCCATGAAGTATTTAACATTTAATAAAAA-AACAG-3′; 5′-AAAAGAATTCTTATCTATGTATGTGAC-AAGACT-3′ (restriction sites for cloning underlined) and P.alciparum K1 strain genomic DNA as template. The PCRroduct was cloned into pGEX-4T1 (GE Healthcare), a plasmidesigned for protein fusions to the glutathione S-transferaseGST) moiety. The integrity of the recombinant PFC0635c cod-ng sequence was determined by automated methods (performedy the Bioservice Unit, BIOTEC, Thailand). The recombinantlasmid was then transformed into BL21(DE3) Escherichia colind expression was induced at 20 ◦C overnight by the additionf 0.4 mM isopropyl-1-thio-�-d-galactopyranoside (IPTG) toulture in Luria Bertani media grown to mid-log phase. Cellsere resuspended in lysis buffer (20 mM MES pH 5.8, 50 mMaCl, 1 mM EDTA, 10% glycerol) and disrupted by Frenchress. Cell extract was clarified by centrifugation for 30 min at0,000 × g and the supernatant was loaded onto a SP-sepharoseGE Healthcare) column equilibrated in lysis buffer. Proteinsere eluted using a linear gradient of NaCl from 50 to 1000 mM

n lysis buffer. Fractions containing recombinant P. falciparumIF4E protein (GST-PfeIF4E) were stored at −80 ◦C.

.2. Cap binding assays

Cap binding activity of GST-PfeIF4E was tested by bind-ng to m7GTP sepharose and UV cross-link assay. For m7GTPepharose binding assay, approximately 859 �g of SP-sepharoseractionated GST-PfeIF4E protein extract, or crude extract fromST-expressing cells transformed with pGEX-4T1 vector were

ncubated with 20 �l of m7GTP sepharose 4B beads (GE Health-are) equilibrated in phosphate buffered saline (PBS) (137 mMaCl, 8 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4).eads were washed four times in 1 ml PBS and then subse-uently washed with 1 ml of PBS containing 1 mM GTP toemove non-specifically bound proteins. Cap-specific proteinsere eluted with 1 ml of PBS containing 1 mM m7GTP. 2.5 �lf each sample were separated by electrophoresis in 12% SDSolyacrylamide gel and electro-blotted onto Hybond ECL mem-

rane (GE healthcare). The blotted proteins were subjected toestern detection with a polyclonal goat anti-GST antibody at a

:10,000 dilution (GE Healthcare) as recommended by the man-facturer. A peroxidase-labelled horse anti-goat IgG antibody

oia1

l Parasitology 155 (2007) 146–155 147

t a 1:10,000 dilution (Vector laboratories) and SuperSignalest pico chemiluminescence (Pierce) were used to detect the

rimary antibody.For UV-link RNA binding assays, radiolabelled, RNA

robe of sequence: 5′-GGGAAUGUAUGUUAAUUGUAUG-AUUA-3′ was synthesized using a T7 maxiscript kit (Ambion)ccording to the manufacturer’s recommendations, together with35S] �-UTP and m7G(5′)ppp(5′) cap analogue (GE Health-are). Template DNA was prepared from annealed partial duplexligonucleotides, following the strategy described in [12].adio-labelled RNA was treated with RQ1 DNaseI (Promega),henol–chloroform extracted and purified with a MicroSpinTM

-25 column (GE Healthcare). RNA binding reactions (10 �l)ontained approximately 1 × 104 cpm of radio-labelled RNA,�g SP-sepharose partially purified GST-PfeIF4E or 1 �g con-

rol crude extract from GST expressing bacteria in binding buffer20 mM HEPES pH 7.5, 40 mM KCl, 10 mM DTT, 10% glyc-rol (v/v), 0.1 mg/ml polyA RNA (GE Healthcare) and 3 mMgCl2). Solutions of m7G(5′)ppp(5′)G or G(5′)ppp(5′)G cap

nalogues for quantitative competition binding reactions wererepared in diethylpyrocabonate-treated water as described byhe manufacturer (GE Healthcare). Binding reactions were incu-ated for 20 min at room temperature, and then exposed toltra-violet irradiation for 15 min in a GS Genelinker (Bio-ad) to cross-link protein/RNA complexes. Reaction productsere separated by electrophoresis in 12% SDS polyacrylamideel. Gels were dried and exposed to a Phosphorimager Generalurpose storage screen (GE Healthcare) and scanned using ayphoon 9410 scanner (GE Healthcare). The band intensitiesf the GST-PfeIF4E/RNA complexes were quantified using themagequant v5.2 program (Molecular Dynamics). Curve-fittingo obtain and statistically compare Kd values was performedsing the un-weighted one site competition model, with sharedaximum and minimum values for all data in the GraphPadrism 4.0 program (GraphPad software).

.3. Purification of P. falciparum capped mRNA withST-PfeIF4E

P. falciparum parasites of strain K1 were cultured at 37 ◦Cn RPMI 1640 medium supplemented with 25 mM HEPES,H 7.4, 0.2% NaHCO3, 40 �g/ml gentamicin, 10% humanerum and human erythrocytes as described [13]. To supportarasite growth, the air was maintained at 3% CO2 auto-atically in a CO2 incubator (Heraeus) [14]. Parasites were

iberated from host cells by saponin-lysis. Total parasite RNAas obtained from asynchronous parasites using Trizol (Invit-

ogen) as recommended by the manufacturer. SP-sepharoseurified protein containing GST-PfeIF4E (approximately 1 mg)as bound to 0.4 ml of glutathione sepharose 4B beads (GEealthcare) equilibrated in PBS. Beads were washed four times

n 10 ml of PBS and then 100 �g of total P. falciparum RNAere incubated with the bead-bound GST-PfeIF4E in 1 ml

f PBS containing 1 mM GTP. After 10 min incubation once, the beads were washed four times in 10 ml PBS. mRNAnd GST-Pf IF4E were eluted from the washed beads usingml of 0.3 M sodium acetate pH5.5 containing 1% SDS. Elu-

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te was phenol-chloroform extracted, isopropanol precipitatednd resuspended in diethylpyrocabonate-treated water. Typicalields were 2–7 �g of mRNA.

.4. Microarray analysis

Microarrays were fabricated by spotting 70-mer oligonu-leotides (30 �M solutions in 3× SSC) on UltraGAPS slidesCorning). The oligonucleotides were composed of the P. fal-iparum optimized set (Operon, version 1.1.1) representing thentire genome and the human control set (Operon, version 4.0).abelled cDNA target was prepared from 20 �g of total P.

alciparum RNA or 2 �g of GST-PfeIF4E purified RNA. TheNA was heated at 70 ◦C for 5 min together with 2.5 �g ofligo-dT(21) and then placed on ice, after which cDNA was syn-hesized by the addition of: dATP, dCTP, dGTP (0.5 mM each);TTP, 0.2 mM; amino allyl-dUTP (Sigma), 0.3 mM; MgCl2,mM; ImpromII reverse-transcriptase 2.5 �l (Promega). Afterh incubation at 37 ◦C, RNA was hydrolysed by adding EDTA

o 100 mM and NaOH to 200 mM and incubating at 65 ◦C for5 min, then neutralized with 25 �l of 1 M HEPES (pH 7.4).DNA samples were desalted on Microcon 30 columns (Milli-ore), and then coupled to 1 pmol monoreactive Cy dyes (GEealthcare), specifically Cy3 to GST-PfeIF4E purified RNA

ample and Cy5 to total RNA sample in 0.1 M sodium bicar-onate buffer (pH 9.0) for 1 h at room temperature in theark. Unincorporated dyes were removed by purification withIAquick PCR purification columns (Qiagen). Labelled cDNAsere quantified by Nanodrop ND1000 (Nanodrop Technolo-ies) and dried down with a vacuum centrifuge. The labelledDNAs were resuspended and combined in appropriate volumesf Pronto! Long Oligo/cDNA hybridization solution (Corning)o give 1 pmol/�l Cy5 and 0.25 pmol/�l Cy3 cDNAs. Microar-ays were hybridized to the Cy3/Cy5 mix overnight at 42 ◦C.fter hybridization, the microarrays were washed according to

he Pronto!Universal Hybridization Kit instructions (Corning)nd scanned using a ScanArray 4000 scanner (Packard Bio-cience).

Spot images were processed and quantified using Scan-lyze software (Eisen, available from: http://rana.lbl.gov/isenSoftware.htm). Bad spots were flagged by visual inspec-

ion of the spot images and cutoff filtering using fractionf pixels in the spot > 1.5 times above the backgroundCH1GTB2 and CH2GTB2 > 0.55). Flagged spots were notonsidered for further analysis. The processed data were nor-alized within each array by the scaled print-tip method, and

etween arrays, using the Aroma package (Bengtsson, avail-ble from: http://www.maths.lth.se/help/R/aroma/) run in Rroject environment (http://cran.r-project.org). The microarrayata have been deposited in NCBIs Gene Expression OmnibusGEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessiblehrough GEO Series accession number GSE6356. Differen-ially expressed genes were identified from the normalized

ata for 5095 array features using significance analysis oficroarrays (SAM). SAM is a non-parametric test that uses

he variation in the dataset to calculate the significance as thealse discovery rate (FDR) [15] (available from: http://www-

GGAc

l Parasitology 155 (2007) 146–155

tat.stanford.edu/∼tibs/SAM/). The thresholds for significanceetermined by SAM were: delta = 0.59, FDR = 0%. The microar-ay data were obtained from four hybridizations using RNArom two independent GST-PfeIF4E purifications from separatealaria cultures.

.5. Northern blotting

P. falciparum total RNA and GST-PfeIF4E purified RNAere mixed with Glyoxal loading dye (Ambion) and heated for0 min at 50 ◦C. The samples were separated by electrophore-is in 1.2% agarose gels in 1× TBE and capillary blottedvernight onto BrightStar Nylon membrane (Ambion) in 10×SC (Ambion). RNA was fixed by UV using a GS GenelinkerBio-rad), washed with 10 mM Tris pH 8.0 and pre-hybridized in.25 M sodium phosphate pH 7.4 containing 7% SDS at 68 ◦C.adio-labelled antisense probes were prepared by in vitro tran-

cription from linearized plasmid template as described above.n order to construct probe templates, full-length gene cod-ng regions for PF13 0141 (Pflactate dehydrogenase, LDH) andFC0635c (PfeIF4E) sequences were obtained by PCR from P.

alciparum genomic DNA and cloned into pGEM3zf(+) plasmidPromega). Membranes were hybridized with probe overnightt 68 ◦C in 25 ml of 0.25 M sodium phosphate pH 7.4 containing% SDS and then washed three times each with 2× SSC, 0.1%DS and then 0.1× SSC 0.1% SDS before phosphorimaging.

.6. PCR quantification of RNA enrichment byST-PfeIF4E

Total P. falciparum and GST-PfeIF4E purified RNA samplesere treated with Turbo DNaseI DNA free (Ambion) to remove

ontaminating genomic DNA. RNA was reverse-transcribedith oligo-dT(21) and ImpromII (Promega) as recommended by

he manufacturer in 50 �l reaction volume containing 340 ng/�lotal RNA or 10 ng/�l GST-PfeIF4E purified RNA. Relativebundances of different RNAs between the two RNA sam-les were determined by real-time PCR using iQSybr Greenupermix (Bio-rad) and an iCycler real-time PCR thermocyclerBio-rad) in 25 �l reactions containing 250 nM of each primernd 1 �l of reverse-transcription reaction template. The PCRrogram used was as follows: 95 ◦C for 3 min; 40 cycles of5 ◦C for 15 s, 58 ◦C for 60 s. Threshold cycle of fluorescenceCt) was determined automatically by the iCycler program (Bio-ad). Mean and standard deviation of Ct were calculated fromminimum of three replicates for each gene. The specificity

f the PCR amplifications was checked by melt-curve analysisnd 4% 3:1 Nusieve:GTG agarose (FMC) gel electrophoresisf the final products. To determine the significance of transcriptnrichment in the GST-PfeIF4E purified RNA samples, one-ailed equal variance t-tests comparing dCt of total and purifiedNAs were performed using Microsoft Excel.

The following primer pairs were used: PFA0310c: 5′-

CTATACCAGAAGGATTGCCAGCAG-3′, 5′-CGGTTGTT-TCATTTGATTTGTTGT-3′ PFE0350c: 5′-GGCTACCATA-GACCTGTTGC-3′, 5′-TTTCCAAAAGCACCTTGACC-3′oxI: 5′-AGATACATGCACGCAACAGG-3′, 5′-AGTTGCA-

emical Parasitology 155 (2007) 146–155 149

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P.J. Shaw et al. / Molecular & Bioch

CCCAATAACTCA-3′ PFE1150w: 5′-CAGCAATCGTTG-AGAAACA-3′, 5′-GCAGATCCAGATTGGTTTGA-3′FC0635c: 5′-TTGGGTGATATGGGAACAAG-3′, 5′-GCT-GATCTTCCCACATAGG-3′ PF13 0141: 5′-GCACCAAAA-CAAAAATCGT-3′, 5′-ACATCTGCTCCAGCCAAATC-3′AL13P1.344: 5′-GCCACTTATTTAGCGGACCA-3′, 5′-GT-TGTTGGCGACTGTGTTT-3′.

.7. 5′-end RNA modifications and RNAmmunoprecipitations

For 5′-end RNA modifications, RNA was treated withither vaccinia virus guanyltransferase (Ambion) to add a7G(5′)ppp(5′)N cap structure to the 5′-end, or tobacco acid

hosphatase (EPICENTRE Biotechnologies) to remove the cap,ollowing the manufacturer’s recommendations. 5 �g of syn-hetic antisense PFC0635c RNA (see above, Section 2.5) was′-capped. The capped synthetic RNA was divided into twond one-half subsequently de-capped with tobacco acid phos-hatase. For experiments with P. falciparum RNA, 20 �g ofotal RNA was subjected to cap-addition or de-capping. EachNA sample (unmodified, cap-added, de-capped) was used in

mmunoprecipitation assays with anti-2,2,7-trimethylguanosinegarose-conjugated mouse mAb antibody (Calbiochem) asescribed in [16]. Briefly, antibody-conjugated beads wereashed with cap binding buffer (25 mM Tris pH 7.5, 0.5 mMTT, 10 mM MgCl2,) and each RNA sample (500 ng syntheticNA, 20 �g P. falciparum RNA) was bound to approximately0 �l washed beads for 20 min on ice. The beads were washedhree times in 0.5 ml cap binding buffer. Bound RNAs wereluted in 0.5 ml 1% SDS 0.2 M NaCl. RNAs were purifiedy phenol:chloroform and isopropanol precipitation. The RNAamples were then DNaseI treated and reverse transcribeds above in Section 2.6. For control experiments with syn-hetic RNA, PCR with PFC0635c specific primers (Section 2.6)as performed on reverse-transcription reaction templates withoTaq polymerase (Promega) as recommended by the manu-

acturer. Reactions with P. falciparum RNA were analysed byuantitative PCR as described above in Section 2.6.

. Results

.1. Cap binding function of P. falciparum eIF4E

A standard assay to demonstrate cap binding function ofIF4E proteins is m7GTP sepharose binding assay, in whichhe m7GTP group mimics the cap structure of mRNA specifi-ally bound by eIF4E [17]. Protein binding assays to m7GTPepharose are shown in Fig. 1. The recombinant P. falci-arum eIF4E protein GST-PfeIF4E was able to bind to m7GTPepharose and could be specifically eluted using m7GTP, but notTP. This result shows that the cap-characteristic N7-methyl

roup is important for binding to GST-PfeIF4E, as describedor eIF4E from other species [3]. By contrast, none of the con-rol GST protein could be bound and subsequently eluted with

7GTP. The cap binding function of GST-PfeIF4E was further

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nd 9: 1 mM m7GTP elution. The flow-through fractions (lanes 2 and 6) wereiluted 10-fold in PBS before loading. The migrations of Broad-range prestainedrotein molecular weight markers (Biorad) are indicated to the right.

ested by a UV cross-linking RNA binding assay (Fig. 2.). Aross-linked complex of protein and capped RNA probe wasbserved only in the reactions with GST-PfeIF4E and not theontrols (Fig. 2A, B). This complex could be competed withxcess m7G(5′)ppp(5′)G cap analogue, with an apparent disso-iation constant (Kd) of 1 �M. The un-methylated cap analogue(5′)ppp(5′)G was significantly (t-test P < 0.0001) much less

fficient in competing this complex, with a Kd of 195 �MFig. 2C). These results concur with the m7GTP sepharose bind-ng assays, highlighting the importance of the N7-methyl groupf the 5′ cap structure for binding to GST-PfeIF4E.

.2. Purification of capped mRNA using P. falciparumIF4E

After demonstrating the mRNA cap binding property ofecombinant P. falciparum eIF4E, we used this protein to purifyull-length capped mRNAs from P. falciparum total RNA.RNAs recovered from bead-bound GST-PfeIF4E were intact,

s shown by denaturing agarose gel electrophoresis (Fig. 3A).he purified mRNA sample had a much lower proportion of ribo-omal RNA, and the larger mRNA species (>3000 nt) appearedo be particularly enriched when compared with total RNA.ince the overall migration pattern of the purified mRNA dif-ered from total RNA, the integrity of the purified mRNA waserified by Northern blotting. Northern blotting for two differ-nt gene transcripts showed that both were enriched and intactn the purified sample, producing the expected 1.6 kb band forfLDH [18] (Fig. 3B) and an approximately 2 kb band consis-

ent with the predicted ORF (684 nt) and 5′-UTR (908 nt) [11]or the PfeIF4E gene PFC0635c (Fig. 3C).

We then used microarrays to compare global transcript abun-ance of P. falciparum total RNA with GST-PfeIF4E purified

150 P.J. Shaw et al. / Molecular & Biochemical Parasitology 155 (2007) 146–155

Table 1Genes more than twofold significantly under-represented in the GST-PfeIF4E purified RNA sample

Oligo IDa Gene nameb Description Score (d)c Average fold change

Organelle-encoded RNAse23986 4 coxI Mitochondrial encoded cytochrome oxidase I 3.82 13.28pclp Clp Plastid encoded Clp protease 3.52 9.67prps12 rps12 Plastid ribosomal protein 12, small subunit 2.60 2.79prpl36 rpl36 Plastid ribosomal protein 36, large subunit 2.57 3.46PtRNA-Thr Plastid tRNA-Thr 2.56 6.89PtRNA-Met Plastid tRNA-Met 2.38 4.12prpl2 rpl2 Plastid ribosomal protein 2, large subunit 2.27 3.79ptufa tufA Plastid encoded homologue of bacterial tufA 2.25 2.82prpl4 rpl4 Plastid ribosomal protein 4, large subunit 2.06 2.69prps5 rps5 Plastid ribosomal protein 5, small subunit 2.06 3.18PtRNA-Pro Plastid tRNA-Pro 1.97 2.79prpl16 rpl16 Plastid ribosomal protein 16, large subunit 1.88 4.00PtRNA-Tyr Plastid tRNA-Tyr 1.83 3.04j667 1 tufA Plastid encoded homologue of bacterial tufA 1.66 2.78PtRNA-Gln Plastid tRNA-Gln 1.64 2.80

Nuclear-encoded RNAsks222 1 PF11 0489 Hypothetical protein 3.35 5.61Z 3 90 PFE1150w pfmdr1 multidrug resistance protein 2.51 2.98a8109 3 PFA0310c SERCA-like PfATP6 2.28 2.44n197 2 PF14 0511 Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase 2.27 2.29n150 93 PF14 0076 Plasmepsin 1 precursor 2.17 2.61m25872 4 MAL13P1.271 Vacuolar ATPase, putative 2.16 2.90f63399 1 MAL8P1.40 RNA-binding protein, putative 2.09 2.63M57785 5 chr13.glimmerm 1053 71% identity to 82% of CAH86998.1: hypothetical protein

PC405219.00.0 [Plasmodium chabaudi]2.06 4.46

e21208 1 PFE0410w Triose or hexose phosphate/phosphate translocator, putative 2.05 2.08i15544 1 PFI0405w Hypothetical protein 2.04 2.11m783 4 MAL13P1.344 RNAse L inhibitor protein, putative 2.03 2.47Z 3 80 PFE1150w pfmdr1 multidrug resistance protein 1.94 2.68ks1030 4 PF11 0414 Hypothetical protein 1.81 2.21Ks117 4 PF11 0145 Glyoxalase I, putative 1.81 2.53ks497 2 PF11 0257 Ethanolamine kinase, putative 1.76 2.59M41776 1 Chr13, unannotated 1.73 4.69e20827 3 PFE0270c DNA repair protein, putative 1.72 2.04n130 46 PF14 0546 Hypothetical protein, conserved 1.60 2.21ks111 4 PF11 0310 Hypothetical protein 1.58 2.09

a Oligo ID identifies each oligo in the DeRisi lab malarial transcriptome database (http://malaria.ucsf.edu/) and PlasmoDB (http://plasmodb.org).

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b Gene name annotated in PlasmoDB.c Relative differences in transcript abundance reported by SAM, based on the

NA. As expected, no significant differences between the twoNA samples were found for the majority of genes, indicat-

ng that the majority of transcripts are capped (Table S1).n the other hand, a subset containing 34 array features

howed consistently high Cy5/Cy3 ratios (≥2.0), which wereound to be significant (FDR 0%) indicating that they arender-represented in the GST-PfeIF4E purified RNA speciesTable 1). Many of these array features represent apicoplastnd mitochondrial organelle-encoded RNAs. Interestingly, inddition to the organelle-encoded RNAs there were also 19 fea-ures representing 18 different nuclear-encoded mRNAs. Theseuclear-encoded transcripts encode proteins with a variety ofossible functions, 11 of which the functions are known, or

an be inferred from homology to proteins in other species.t is noteworthy that within this group of nuclear genes, therere three genes of the ATP binding cassette (ABC) trans-orter family, namely pfmdr1 (PFE1150w), RNaseL inhibitor

vfwR

of change in abundance and standard deviation.

MAL13P1.344) and SERCA-like PfATP6 (PFA0310c). Otherotable genes in this group include: glucose-6-phosphateehydrogenase-6-phosphogluconolactonase (PF14 0511), gly-xalase I (PF11 0145) and putative DNA repair proteinPFE0270c).

To verify the microarray results, quantitative PCR waserformed comparing transcript abundance in total RNAnd GST-PfeIF4E purified RNA for seven genes, chosen toepresent transcripts found by microarray analysis to be sig-ificantly (coxI, PFE1150w, PFA0310c and MAL13P1.344)r not significantly under-represented (PFE0350c, PF13 0141,nd PFC0635c) in the GST-PfeIF4E purified sample (Fig. 4).he degree of enrichment measured by quantitative PCR

aried for each transcript, from 0.9 (PFE1150w) to 22.1-old (PF13 0141). Highly significant enrichment (P < 0.001)as observed only for the PF13 0141, and PFE0350cNAs, suggesting that the other transcripts studied in this

P.J. Shaw et al. / Molecular & Biochemical Parasitology 155 (2007) 146–155 151

Fig. 2. Cross-link RNA binding assays. (A) and (B) Representative imagesof UV cross-link RNA binding assays. Lane 1: control, no protein added;lane 2: control, crude extract from bacteria expressing GST protein; lane3: GST-PfeIF4E protein; lanes 4–9: GST-PfeIF4E RNA binding competedwith 10-fold dilutions of cap analogue, G(5′)ppp(5′)G shown in (A) andm7G(5′)ppp(5′)G in (B). Competitor concentrations varied from 1 (lane 4) to10 nM (lane 9). The migration positions of GST-PfeIF4E/RNA cross-linkedcomplex and unlinked RNA probe are marked. (C) Quantitative analysis of GST-PfeIF4E/RNA cross-linking competed with cap analogues: m7G(5′)ppp(5′)G(tc

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Fig. 3. Purification of 5′-capped P. falciparum RNA. (A) 1.2% denaturingagarose gel. Lane M: millenium RNA marker (Ambion); lanes T6, T3, T1.5: 6,3, and 1.5 �g of P. falciparum total RNA, respectively; lane C: 1.5 �g of GST-PfeIF4E purified RNA. (B) Northern blot of gel in (A) probed with PF13 0141(PfLDH gene) probe. (C) Northern blot of gel in (A) probed with PFC0635c(PfeIF4E gene) probe.

Fig. 4. Quantitative PCR comparing transcript abundance in total and GST-PfeIF4E purified RNA. Mean threshold cycles normalized to coxI (dCt) forboth total RNA (white bars) and GST-PfeIF4E purified RNA (black bars) sam-ples are plotted for each gene (shown by PlasmoDB ID along the horizontalaxis). For gene descriptions, see text and Table 1. Error bars represent stan-dard deviations. The fold enrichment in GST-PfeIF4E purified RNA relative

squares) and G(5′)ppp(5′)G (triangles). The data points are the mean from 3o 7 replicates and the error bars show the standard deviations. The dissociationonstants (Kd) are calculated from curve fitting to the data (see Section 2).

xperiment are inefficiently enriched by GST-PfeIF4E purifi-ation.

.3. Probing the 5′-end structures of putatively uncappedNAs

The lack of enrichment for some RNAs by eIF4E implieshat they do not have cap structures, either because the cap isot added, as in the case of the organelle-encoded RNAs, orhe cap has been removed (see Section 4). Alternatively, fea-ures inherent to these transcripts (e.g. secondary structure) maynterfere with eIF4E recognition. A more detailed investigationf the cap status was performed on transcripts representativef putatively predominantly capped PfLDH (PF13 0141) and

ncapped pfmdr1 (PFE1150w) RNAs, for which detailed 5′ tran-cript mapping data are available. For the PfLDH (PF13 0141)ene, several 5′ transcription start sites within a short clusterave been mapped by the cap-dependent oligo-capping method

to total RNA is shown underneath each gene. Fold-enrichment is calculatedas 2−(ddCt), where ddCt is the difference in dCt between total and purifiedRNA samples. P values for GST-PfeIF4E enrichment: PFE1150w, 0.410; coxI,0.500; PFA0310c, 0.015; MAL13P1.344, 0.004; PFC0635c, 0.006; PFE0350c,2 × 10−5; PF13 0141, 4 × 10−4.

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11]. By contrast, the pfmdr1 (PFE1150w) transcript has a sin-le start site, mapped 1.94 kb upstream of the pfmdr1 proteinoding region by methods not dependent on a 5′ cap [19].

To further validate our results showing the presence ofncapped RNAs, an alternative method to select capped RNAas used. Immunoprecipitations of capped RNAs were per-

ormed with an antibody known to recognize 7-methylguanosineapped mRNA. To validate the specificity of this antibody,mmunoprecipitation assays were performed with a syntheticNA (Fig. 5A). The 5′ end of this RNA was modified by theddition of a cap (capped sample) and subsequent cap removalrom capped RNA (de-capped sample). Of the immunoprecipi-ated eluate samples, the synthetic RNA is clearly detected onlyn the capped RNA sample. Faint bands can be seen in thencapped and de-capped RNA samples, however, suggesting

hat the antibody does not recognize capped RNA exclusively.mmunoprecipitations of P. falciparum RNA were performed,nd the abundances of the PfLDH (PF13 0141) and pfmdr1PFE1150w) transcripts measured (Fig. 5B). For unmodified,

ig. 5. Immunoprecipitation assays with anti-2,2,7-trimethylguanosine anti-ody. (A) Control immunoprecipitation with synthetic RNA. PCR products wereeparated in 2% agarose gel. Lane NT: no template; lanes marked U: immuno-recipitation with uncapped RNA; lanes marked C: immunoprecipitation with′-capped RNA; lanes marked D: immunoprecipitation with De-capped RNA.anes marked −: no RT enzyme added; Lanes marked +: RT enzyme added.he far-left hand lane is DNA marker pUC19 HpaII (Ambion). (B) QuantitativeCR data from immunoprecipitations with P. falciparum RNA. The differ-nce in mean threshold cycles (dCt) between pfmdr1 (PFE1150w) and PfLDHPF13 0141) are plotted for flow-through (white bars) and immunoprecipitationluate RNA samples (black bars). Immunoprecipitations were performed withnmodified, cap-added and de-capped P. falciparum RNA. Error bars representtandard deviations. The fold-difference in flow-through relative to immunopre-ipitated eluate sample is shown underneath. Fold-difference is calculated as−(ddCt), where ddCt is the difference in dCt between flow-through and eluateamples.

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otal P. falciparum RNA, the PfLDH (PF13 0141) transcript ispproximately 10-fold more abundant than pfmdr1 (PFE1150w)n the immunoprecipated eluate relative to the flow-through.

odification of P. falciparum RNA by cap addition or de-apping resulted in a reduction in the difference in abundanceetween the PfLDH (PF13 0141) and pfmdr1 (PFE1150w)ranscripts in the immunoprecipated eluate relative to the flow-hrough. The reduction was more marked for de-capped P.alciparum RNA.

. Discussion

In this study, we have functionally characterized a P. fal-iparum eIF4E homologue. P. falciparum eIF4E recombinantrotein (GST-PfeIF4E) bound to m7GTP sepharose and waspecifically eluted by m7GTP (Fig. 1). GST-PfeIF4E was showno preferentially bind the m7G(5′)ppp(5′)G cap analogue inross-link RNA binding assay (Fig. 2). These results confirmxpectation that P. falciparum eIF4E has RNA cap bindingctivity. The affinity of GST-PfeIF4E for the m7G(5′)ppp(5′)Gap analogue, inferred from competition assays, is similar tohe values reported for recombinant GST-eIF4E proteins fromther species [20]. Results from Northern blotting and microar-ay analyses comparing total and GST-PfeIF4E purified RNAsonfirmed the practical use of this protein for purifying P. falci-arum 5′-capped mRNA, as has been demonstrated previouslyor mammalian eIF4E [21,22]. Comparison of transcript abun-ance between total and GST-PfeIF4E purified P. falciparumNA by microarray analysis (Table 1) and quantitative PCR

Fig. 4) indicated that several transcripts are poorly enriched byST-PfeIF4E purification. The poorly enriched transcripts areutatively uncapped RNAs.

.1. Uncapped P. falciparum RNA

Amongst the poorly enriched transcript subset in GST-feIF4E purified RNA are genes from the organelle genomes

apicoplast and mitochondrion). These transcripts are notxpected to be enriched by GST-PfeIF4E, since they are notynthesized in the nucleus where capping occurs, and are there-ore not capped. Unexpectedly, we also found transcripts fromeveral nuclear genes in the poorly enriched transcript subset. Toest whether cap selection with GST-PfeIF4E is inefficient forome RNAs because of some technical detail specific to GST-feIF4E, alternative cap-selection methods were investigated.he cap-trapper method was first tested, in which a biotin group

or purification is introduced onto the cap [23]. The enrichmentor capped RNA with this method was however, unsatisfactorydata not shown). Selection of capped RNA by immunoprecipita-ion was tested, and cap-selectivity demonstrated (Fig. 5A). ThefLDH (PF13 0141) transcript was found to be relatively morebundant in the cap selected immunoprecipitated sample fromotal P. falciparum RNA than pfmdr1 (PFE1150w) (Fig. 5B).

his result is in agreement with our other data from experimentserformed with GST-PfeIF4E. In our hands, capped RNA purifi-ation using recombinant eIF4E is superior to other methods, ingreement with other groups [24].

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The under-represented nuclear transcripts from cap-selectionay lack a cap, or the cap is inaccessible to the eIF4E protein

ecause of RNA secondary structure immediately adjacent to theap. To prove whether these mRNAs actually lack a cap, enzy-atic cap modifications of P. falciparum RNA were performed

Fig. 5B). Cap modification (both addition and removal) resultedn reduced abundance of the PfLDH (PF13 0141) transcript rel-tive to pfmdr1 (PFE1150w) in cap selected RNA. We inferrom the result of cap-addition that if a significant proportion ofhe pfmdr1 (PFE1150w) RNA has uncapped 5′ ends to which aap can be added, relatively more of this transcript can be capelected after cap addition. Moreover, we infer from the resultith de-capped P. falciparum RNA that de-capping adversely

ffects the efficiency of cap-selection of PfLDH (PF13 0141)ore than pfmdr1 (PFE1150w), if the former transcript ini-

ially has a greater proportion of caps than the latter. These datahus support the notion that there are uncapped P. falciparum

RNAs. Although secondary structure cannot be discounted asfactor for inefficient cap-selection of the pfmdr1 (PFE1150w)

ranscript, we think this is of minor importance, as secondarytructure prediction does not indicate the presence of stabletructure in the immediate vicinity of the 5′ end of this RNA (nothown), and no secondary structure impediment was reported forts transcript mapping [19].

The uncapped nuclear P. falciparum mRNAs may have failedo undergo capping after transcription, although this is unlikelyiven the tight linkage between capping and transcription in theucleus [2]. Since cap removal is part of the natural cycle ofukaryotic mRNA turnover, it is more likely that the uncappeduclear P. falciparum mRNAs are de-capped mRNAs. The cap isemoved by the de-capping enzyme Dcp1p in cytoplasmic foci,alled processing (P-) bodies, and the RNA is degraded [9].ertain mRNAs in P-bodies may also be stored (translationallyuiescent) and later exit the P-bodies to be translationally activegain [25].

.2. Uncapped mRNA function with respect to cellulartress

It is of interest that in the subset of predominantly uncappeduclear-encoded transcripts there are genes with functions impli-ated in response to cellular stress. The glucose-6-phosphateehydrogenase-6-phosphogluconolactonase (PF14 0511) andlyoxalaseI (PF11 0145) genes have roles in anti-oxidant detox-fication, since the former gene function performs the first twoteps in the pentose phosphate pathway, generating NADPHequired for reduction of reactive oxygen species and the lat-er gene function detoxifies oxidant damage on DNA, RNAnd proteins [29]. The putative DNA repair protein PFE0270cay be important for repairing oxidant-induced DNA dam-

ge. If exposure to anti-malarial drugs can be considereds cellular stress, then the functions of the uncapped tran-cripts PFE1150w, PFA0310c and MAL13P1.344 should be

ecognized. These genes have been implicated as drug tar-ets, or mediators of drug resistance, one of which (pfmdr1)as been well-documented. Changes in pfmdr1 (PFE1150w)ene copy number, combined with point mutations, modulate

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l Parasitology 155 (2007) 146–155 153

rug resistance to common antimalarial drugs such as qui-ine, mefloquine and artemisinin [26]. SERCA-like PfATP6PFA0310c) has been shown to be a target for the antimalarialrug artemisinin [27]. The RNaseL inhibitor (MAL13P1.344)ene expression is out of phase between the mefloquine drugensitive HB3 strain compared with the mefloquine resis-ant strains 3D7 and Dd2, suggesting that subtle changes inxpression of this gene could contribute to drug-resistance28].

The pfmdr1 (PFE1150w) gene has a 1.94 kb long 5′ tran-cribed sequence before the putative protein-coding region [19]ontaining 11 upstream open reading frames (uORFs). The mul-iple uORFs suggest that canonical cap-dependent translationf pfmdr1 (PFE1150w) would be inefficient, since multiple re-nitiations of the ribosome or “leaky” scanning of the uORFs30] would be required for translation. It has been shown thatome mRNAs with long 5′ flanking regions and multiple uORFsan be translated by cap- and eIF4E-independent mechanisms.hese mechanisms are dependent on sequence/structural fea-

ures in the 5′ regions of the mRNA, such as internal ribosomentry sites (IRES) [30].

It remains to be investigated whether uncapped P. fal-iparum transcripts such as pfmdr1 (PFE1150w) possessap-independent translation elements. These elements couldnable the parasite to selectively synthesize these proteins fromtored, de-capped transcripts under stress conditions, such asrug challenge or oxidant stress. In this respect, the parasiteould possess a stress survival mechanism similar to other

pecies such as yeast, where it has been shown that several genesre translationally up-regulated in response to oxidant stress.hese up-regulated genes include several with anti-oxidantnzyme and ABC transporter protein functions [31]. In mam-alian cells, selective translation by eIF4E/cap-independent

xpression is induced for several apoptotic response genes fol-owing cellular stress [32].

.3. Uncapped transcripts and transcript storage

For some of the putatively uncapped P. falciparum mRNAse found, there are reports of post-transcriptional regulation,hich can be linked with de-capped RNA. Post-transcriptionalene regulation has been described for the Plasmodium bergheiurine malarial parasite, in which several transcripts with

unctions important in the mosquito stages are known to beranslationally repressed in the gametocyte stage. This repres-ion is mediated by a DDX6 RNA helicase, development ofygote inhibited (DOZI) [33] DOZI shares homology with theeast Dhhp1 protein, which recruits the mRNA de-cappingnzyme Dcp1p [34]. It is thought that DOZI recruits tran-cripts with distinct 3′ UTR RNA regulatory elements, whichre partially conserved among Plasmodium species [35] toranslationally repressed complexes. Also in that study [33],t was found that the P. berghei homologue of the pfmdr1

PFE1150w) transcript is down-regulated together with knownranslationally quiescent transcripts in gametocyte-stage DOZI

utant parasites. Therefore, the Plasmodium mdr1 RNA iserhaps normally targeted by DOZI for storage through-

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ut the life cycle, leading to accumulation of de-cappedranslationally quiescent transcripts. In agreement with thisonjecture, the protein expressions from the P. berghei homo-ogues of the P. falciparum pfmdr1 (PFE1150w), putativeNA repair protein (PFE0270c) and putative vacuolar ATPase

MAL13P1.271) genes we found with predominantly uncappedranscripts are negatively correlated with DOZI protein lev-ls, when comparing between male and female gametocytes36].

In conclusion, it has been demonstrated that P. falciparumas a canonical eIF4E which interacts with capped mRNA.

class of transcripts was described which are predominantlyncapped. These transcripts are possibly targeted for storagend cap removal, which may have implications for the parasite’sesponse to stress, including antimalarial drugs.

cknowledgements

We thank Prof. Camilla M. Kao (Stanford Uni., USA) foricroarray printing facilities, Ms. Jarunee Vanichtanankul for

indly providing us the PfLDH plasmid clone, Ms. Mayurachatoopha for culturing parasites, Dr. Curt Hagedorn for advice onecombinant eIF4E purification and Prof. Prapon Wilairat forritical review of the manuscript. This work receives supportn part from the Welcome Trust, Medicines for Malaria Ven-ure (MMV) Programmes to Y.Y. and UNICEF/UNDP/Worldank/WHO Special Programme on Tropical Diseases to Y.Y.nd S.K. Support from Thailand Tropical Diseases Research (T-) Programme to S.K. and P.J.S. is also gratefully acknowledged..K. is an international scholar of the Howard Hughes Medicalnstitute (HHMI, USA).

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at doi:10.1016/j.molbiopara.2007.07.003.

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