Biochimica et Biophysica Acta 1819 (2012) 1186–1199

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Biochimica et Biophysica Acta

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Plasmodium falciparum Prp16 homologue and its role in splicing

Prashant Kumar Singh a, Shivani Kanodia a, Chethan Jambanna Dandin b,Usha Vijayraghavan b, Pawan Malhotra a,⁎a Malaria Research Group, International Centre for Genetic Engineering and Biotechnology, New Delhi 110067, Indiab Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India

⁎ Corresponding author at: International CentreBiotechnology, Aruna Asaf Ali Marg, NewDelhi 110067, Indfax: +91 11 26162316.

E-mail address: pawanm@icgeb.res.in (P. Malhotra)

1874-9399/$ – see front matter © 2012 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.bbagrm.2012.08.014

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 May 2012Received in revised form 29 August 2012Accepted 30 August 2012Available online 7 September 2012

Keywords:MalariaParasiteHelicasePrp16Splicing

Large numbers of Plasmodium genes have been predicted to have introns. However, little information existson the splicing mechanisms in this organism. Here, we describe the DExD/DExH-box containing Pre-mRNAprocessing proteins (Prps), PfPrp2p, PfPrp5p, PfPrp16p, PfPrp22p, PfPrp28p, PfPrp43p and PfBrr2p, presentin the Plasmodium falciparum genome and characterized the role of one of these factors, PfPrp16p. It is amember ofDEAH-box protein family with nine collinear sequence motifs, a characteristic of helicase proteins. Experimentswith the recombinantly expressed and purified PfPrp16 helicase domain revealed binding to RNA, hydrolysis ofATP as well as catalytic helicase activities. Expression of helicase domain with the C-terminal helicase-associateddomain (HA2) reduced these activities considerably, indicating that the helicase-associated domainmay regulatethe PfPrp16 function. Localization studies with the PfPrp16 GFP transgenic lines suggested a role of its N‐terminaldomain (1–80 amino acids) in nuclear targeting. Immunodepletion of PfPrp16p, from nuclear extracts of parasitecultures, blocked the second catalytic step of an in vitro constituted splicing reaction suggesting a role for PfPrp16pin splicing catalysis. Further we show by complementation assay in yeast that a chimeric yeast-Plasmodium Prp16protein, not the full length PfPrp16, can rescue the yeast prp16 temperature‐sensitivemutant. These results suggestthat although the role of Prp16p in catalytic step II is highly conserved among Plasmodium, human and yeast, subtledifferences exist with regards to its associated factors or its assembly with spliceosomes.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Intron removal during pre-mRNA splicing is a complex yet highlyprecise and essential process during gene expression in metazoan aswell as protozoan species. Pre-mRNA splicing requires an ordered as-sembly of U1, U2, U4/U6.U5 RNAs and transacting non-snRNP proteinsinto dynamic complexes referred as spliceosomes [1]. Spliceosome as-sembly starts with the sequential binding of U1 and U2 snRNPs topre-mRNA to form pre-spliceosome which is further converted intopre-catalytic spliceosome by the recruitment of U4/U6.U5 tri-snRNP.Subsequently, the pre-catalytic spliceosomeundergoesmajor structuraland compositional re-arrangements marked by the dissociation of U1and U4 snRNPs that gives rise to activated spliceosome. After activa-tion, the spliceosome undergoes further conformational switches toform catalytically active spliceosome which catalyzes the splicingof the pre-mRNA. Catalysis occurs by two sequential trans-esterification

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reactions; the first step involves cleavage at the 5′ splice site and forma-tion of a 2′–5′ branched intermediate (lariat–exon), while in the secondstep the 3′ splice site is cleaved and the exons are ligated [2,3]. Eachstep of spliceosome assembly, except recruitment of U1 snRNP onpre-mRNA, occurs in ATP dependent manner [3]. During first and sec-ond trans-esterification reactions, initial conformational changes ofspliceosome are ATP dependent and subsequent formation of lariat in-termediates and mature message are ATP independent steps [4]. Uptill now, the spliceosomal complexes and their associated proteinshave beenwell described in Trypanosome, yeast and human [2,3,5]. Try-panosomes are particularly interesting as they show both trans as wellas cis splicing and exhibit variation in Sm core structure [6]. Little infor-mation is available on the splicing machinery and its components inother organisms.

Spliceosome is essentially a protein–RNA complex and nearly two-thirds of themass of the human spliceosome is of snRNP associated pro-teins. In addition,multiple non-snRNP proteins are also involved in splic-ing. Mass spectrometry based studies on purified yeast and humanspliceosomes have identified ~80 to ~170 splicing related proteins re-spectively [7]. Many yeast pre-mRNA processing proteins (Prps) wereuncovered by screens of conditional lethal mutants for splicing defectsand by the isolation of second site suppressors ofmutants in spliceosomalsnRNAs [8,9]. One class of yeast factors, Prp2p, Prp5p, Prp16p, Prp22p,

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Prp28p, Prp43p and Brr2p, is of particular interest in light of the re-quirement of ATP hydrolysis in splicing [2,3]. These factors belongto DExD/DExH-box family of proteins and remodel the spliceosomefacilitating multiple yet sequential transitions between mutuallyexclusive RNA-RNP interaction networks [2,3]. DExD/DExH-box splicingfactors are characterized by a helicase domain, a non-conservedN-terminal region and a C-terminal region which is conserved onlyamongst a subset of these proteins (Prp2p, Prp16p, Prp22p andPrp43p). Even though all of these splicing factors share the helicasedomain, subdivided in to N-terminal DEAD region and a C-terminalHELICASE_C region (Pfam database) [10], each factor acts at preciselydefined steps of splicing in vitro and in vivo [2,3]. Prp5p and Prp28pfunction early in spliceosome assembly whereas Brr2p functions areneeded both during assembly and disassembly of spliceosomes. Prp2p-mediated ATPase activity promotes the transformation of activatedspliceosome into the catalytically active spliceosome. Prp22p has dualfunctions; it associates with catalytically active spliceosomes to aid thesecond step and it is also involved in mRNA release from spliceosomes.Despite these specific roles, the recruitment of these ATPases during as-sembly and splicing is not mutually exclusive as presence of Prp43p hasbeen observed from early steps of spliceosome assembly to complex dis-assembly where it finally acts [2,3]. It is speculated that the temporal ac-tivity and functional specificity of the Prps is determined by their non-conserved domains.

Among the different Prps identified, Prp16p has been well stud-ied in yeast. The prp16-1 mutant allele was initially identified as adominant suppressor of a branch point mutation (A to C) whichcauses accumulation of un-spliced pre-mRNA indicating the role ofPrp16p in maintaining the splicing fidelity [8]. Later, Prp16p wasalso identified as the temperature‐sensitive mutant prp23-1 thatblocked the splicing of wild type pre-mRNAs and led to the accumu-lation of lariat intermediates [9]. Subsequently it was demonstratedthat the conformational rearrangement of spliceosomes after firstcatalytic step and during second trans-esterification reaction is depen-dent upon theATPase activity of Prp16p [11]. To promote exon–exon liga-tion, during the second step, these conformational changes are achievedby destabilizing RNA-RNA interaction of U2 and U6 snRNAs with thebranchpoint site and the 5′ splice site respectively to facilitate the realign-ment of splice site in the lariat intermediate to the 3′ of exon1 [2,3].Prp16p activity has also been linked to the destabilization of U2–U6 du-plex after first catalytic step [12–14].

In Plasmodium falciparum, a human malaria parasite, splicing andsplicingmachinery are poorly characterized. Although anumber of recentstudies have identified Plasmodium helicases, the role of many of thesehelicases in splicing is yet to be elucidated [15]. In the present study,we identified DExD/DExH-box containing splicing factors of Plasmodiumfalciparum, Prp2p, Prp16p, Prp43p, Prp5p, Prp22p, Prp28p, and PfBrr2p,by computational approaches. Amongst these, biochemical properties ofPrp16p proteins and their role in splicing have been well documentedin model organisms and understanding its function can provide a basicframework for understanding the splicing processes in other less studiedhuman parasitic organisms. As an initial attempt towards understandingthe splicing machinery of malaria parasite, PfPrp16 was chosen for fur-ther functional characterization.

Our results showed that PfPrp16p retains all the biochemicalfunctions of its homologues like nucleic acid binding, ATP hydrolysisand nucleic acid unwinding activities and it acts at the second trans-esterification step of splicing. Notably, we found that the helicase andthe C-terminal helicase-associated domain (PfPrphe+ha), when expressedtogether, exhibited considerably reduced ATP hydrolysis as well as RNAunwinding activity suggesting a role for the HA2 domain in regulatingthe activity of PfPrp16p. A chimeric expression construct carrying atranslational fusion of the yeast N-terminal coding sequences with therest of the Plasmodium PfPrp16 coding sequences was able to rescuethe yeast prp16 conditional mutant strain. These results are the firstfunctional demonstrations of a Plasmodium splicing factor and reveal

that the splicing functions of Prp16p are quite conserved from protozo-an to mammals.

2. Materials and methods

Sequences of the primers used in the study are provided in Supp.Table 1. Sequences of the probes for northern analysis are providedin Supp. Table 2.

2.1. P. falciparum culture, plasmid constructs and parasite transfection

P. falciparum strain 3D7 was cultured in human O+ erythrocytesat 37 °C under 5% O2, 5% CO2, and 90% N2 in RPMI 1640 medium(GIBCO BRL) supplemented with 27 mM sodium bicarbonate, 11 mMglucose, 10 mg/mL gentamicin, and 0.5% albumax. Synchronization ofthe parasites in culturewas achieved by sorbitol treatment. To generatetransfection vector constructs 1–120 nt (PfPrp16.1-40-GFP), 1–240 nt(PfPrp16.1-80-GFP), and 241–3456 nt (PfPrp16.81-last-GFP) of PfPrp16were amplified from P. falciparum 3D7 cDNA using PfPrp16-1 and 2, 1and 3, 4 and 5 primers respectively. Amplicons were purified andcloned in frame with the 3′ appended GFP sequence in the BglII andAvrII sites of the pSSPf2 transfection vector [16]. P. falciparum 3D7 par-asites were transfected with 100 μg of plasmid DNA (Plasmid Maxikit, Qiagen) by electroporation (310 V, 950 μF) and the transfected par-asites were selected on 2.5 nMWR99210 drug.

2.2. Identification and sequence features of P. falciparum homologue ofYeast Prp16

Protein sequences of yeast Prp2p, Prp5p, Prp16p, Prp22p, Prp28p,Prp43p and Brr2p were used to search the P. falciparum protein data-base (www.plasmodb.org) using blastp program and sequences withmaximum identity and similarity were downloaded. To further as-certain true homologues, domain-wise alignments were performedin addition to global alignments. Domain architecture and bound-aries were defined by Pfam searches of each protein at Pfam database(www.pfam.sanger.ac.uk) [10]. Nuclear localization signal of PfPrp16pwas predicted using PSORT program (http://www.psort.org). Alignmentof PfPrp16pwith other homologueswas done as essentially described byWu-Scharf et al. [17]. Protein sequences of Schizosaccharomyces pombe(CAB52799), Homo sapiens (AAC27431), Arabidopsis thaliana (Q38953),C. elegans (P34498) and a trans-gene silencing relatedDEAH-box helicaseof Chlamydomonas reinhardti (AF305070)were retrieved fromNCBI data-base and were aligned with PfPrp16p and yPrp16p. Orthologs in otherPlasmodium spp. were identified from PlasmoDB using PfPrp16p se-quence as a query.

2.3. Molecular cloning of PfPrp16 full length gene

For molecular cloning of PfPrp16, total RNA was isolated from asyn-chronous P. falciparum 3D7 cultures, and reverse transcription wasperformed using PfPrp16-6 primer. Complementary DNA was used asa template for PCR reactions, using PfPrp16-6 and PfPrp16-7 primers.Amplicon was cloned into pGEMT easy vector and sequenced by di-deoxy sequencing reaction.

2.4. Semi-quantitative RT-PCR of PfPrp16

Total RNAs were isolated from ring, trophozoite and schizontstages of P. falciparum 3D7 using mini RNA isolation kit accordingtomanufacturer's instruction (Qiagen). Isolated RNAwas used to synthe-size cDNA for each stage using Superscript II first-strand synthesis sys-tem from Invitrogen (Carlsbad, CA, USA). Semi‐quantitative PCR wasperformed for 18 cycles using gene-specific primers. Genomic equiva-lents for each parasite stage were normalized by analysing the expres-sion of α-tubulin gene for all the RNA samples.

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2.5. Expression of recombinant proteins

Three different regions of PfPrp16p, prehelicase, helicase andhelicase-HA2 (PfPrp16ph, PfPrp16he and PfPrp16he+ha), were PCRamplified from P. falciparum 3D7 cDNA library using Pfprp16-8 and 9, PfPrp16- 10 and 11, PfPrp16- 10 and 12 primers respective-ly. The amplified fragments were digested with restriction enzymesBamHI and Sal1 and cloned into corresponding sites of the E. coli ex-pression vector pQE30 (Qiagen, Germany). All DNA sequences wereverified by sequencing. The resultant plasmids were expressed inEscherichia coli M15 cells as 6XHis-tagged proteins. Briefly,transformed M15 cells were grown in LB medium containing kana-mycin (25μg/ml) and ampicillin (100 μg/ml) at 37 °C with constantshaking (200 rpm). When cultures were in mid-logarithmic phase,expression of proteins was induced by 1 mM IPTG at 37 °C for3–4 h and the E. coli cells were harvested by centrifugation. Expres-sion of recombinant proteins was analyzed by coomassie stainingand western blots.

To purify the recombinant proteins, harvested bacterial cells weresubjected to three freeze/thaw cycles and subsequently suspended inlysis buffer (20 mM Tris–HCl, pH 8.0, 250 mM NaCl, 1% Triton, 0.5%Tween 20 and protease cocktail inhibitor). Bacterial cells were furtherdisrupted by sonication and cleared by centrifugation. For recombinantproteins PfPrphe and PfPrphe+ha purification, clear supernatants wereallowed to bind to pre-equilibrated Ni-NTA (Qiagen, GmbH, Germany)in binding buffer (20 mM Tris–HCl, pH 8.0, 250 mM NaCl) for 6 h at4 °C followed by extensive washing with binding buffer supplementedwith 20 mM imidazole. Proteins were finally eluted with a gradient of50–400 mM imidazole in elution buffer (20 mM Tris–HCl, pH 8.0,250 mMNaCl, 10% glycerol and protease inhibitor cocktail). The elutedproteins were further dialyzed against the same buffer without imidaz-ole. For the purification of PfPrp16ph recombinant protein, inclusionbodies were obtained and solubilized in lysis buffer containing 6 Mguanidine hydrochloride. Contents were centrifuged after solubiliza-tion and supernatant was allowed to bind to pre-equilibrated Ni-NTA(Qiagen, GmbH, Germany) in binding buffer (6 M GuHCL, 150 mMNaCl, 10 mM imidazole 20 mM Tris–HCl pH 8.0) at room tempera-ture. Column was extensively washed with binding buffersupplemented with 20 mM imidazole. Elution was carried out byusing a gradient of 50–400 mM imidazole in elution buffer (8 Murea, 20 mM Tris–HCl, pH 8.0, 250 mMNaCl, 10% glycerol and prote-ase inhibitor cocktail). The protein was refolded by dialyzing itagainst decreasing concentration of urea (8 M to 1 M) in elutionbuffer. The purity of all the three recombinant proteins was analyzedon 10% SDS-PAGE.

2.6. Preparation of P. falciparum nuclear and cytoplasmic fractions

P. falciparum nuclear lysate was prepared as described by Lanzeret al. [18]. Briefly, asynchronous culture of P. falciparumwas saponinpurified and washed thrice with ice cold PBS. Parasite pellet was in-cubated for 5 min in ice cold lysis buffer (20 mm HEPES pH 7.8,10 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 1% NP40) andcentrifuged at 2500g for 10 min at 4 °C. Supernatant was aliquotedand stored at −80 °C as cytoplasmic fraction. Pellet was washedtwice with lysis buffer (2500 g, 4 °C) and re-suspended in 1–2 pel-let volume of extraction buffer (20 mM HEPES, pH 7.8, 0.4 M NaCl,1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF). Suspensionwas incubated on ice for 15 min with vigorous shaking after every5 min and then centrifuged at 13,000g for 30 min at 4 °C. Superna-tant (nuclear lysate) was dialyzed against dialysis buffer (20 mMHEPES, 0.2 mM EDTA, 50 mM KCL, 20% Glycerol, 0.5 mM DTT, 2 mMPMSF) in nuclease free environment at 4 °C. Protease inhibitor cocktailwas added in dialyzed nuclear extract. Nuclear extract was aliquotedand stored at−80 °C.

2.7. Western blot analysis

Polyclonal antibodies were raised in rabbit against purifiedPfPrp16ph recombinant protein, a region in PfPrp16p with no ho-mology with any other P. falciparum protein. For immunoblottingexperiments, parasites were released from infected erythrocytesby 0.1% (w/v) saponin treatment. Cell-free protein extractswere prepared by suspending parasite pellets in a buffercontaining 10 mM Tris pH 7.5, 100 mM NaCl, 5 mM EDTA, 1%Triton X-100, and 1× Complete Protease inhibitor mixture(Roche) using a syringe and a needle. Lysates were cleared bycentrifugation at 14,000g for 30 min at 4 °C. After separation oflysate proteins on SDS-PAGE gels, proteins were transferred toa nitrocellulose membrane. Immunoblotting was performedusing the above described anti-PfPrp16p antiserum and horse-radish peroxidase-labelled anti-rabbit IgG. ECL substrate (Pierce)was used to develop the blots following the manufacturer's instruc-tions. For sub-cellular distribution of PfPrp16p equal amounts of cy-toplasmic and nuclear fractions of all three stages were separated onSDS-PAGE gels and immunoblotting was performed as describedabove.

2.8. Indirect immunofluorescence assay and co-localization

Indirect immunofluorescence assayswere performedon P. falciparum3D7 and transgenic parasite lines as described earlier. Briefly, thinsmears of infected erythrocytes were made on a glass slide andfixed with mixture of methanol/acetone. Slides were blocked inblocking buffer (1× PBS, 10% FCS) for 1 h at 37 °C. After blocking,slides were washed thrice with PBS and were incubated with pri-mary antibodies diluted in blocking buffer (mice anti-PfTSN anti-body; dilution 1:100 and rabbit anti-PfPrp16p/anti-GFP antibody;dilution 1:200) for 1 h at 37 °C. After incubation with primary antibody,slideswerewashedwith 1× PBS and incubatedwith anti-mice antibody(conjugated to FITC, dilution 1:100) and anti-rabbit antibody (conjugat-ed to Cy3, dilution 1:300) for 1 h at 37 °C. Finally, the slides werestained with DAPI (final concentration of 2 μg/ml) for 20 min at 37 °C,washed twice in 1× PBS containing 0.05% Tween-20, once in 1× PBSand mounted on a cover slip in the presence of anti fade reagent(BIO-RAD). The slideswere viewedusingNikon TE 2000-U fluorescencemicroscope.

For GFP fluorescence detection, P. falciparum cultures expressingGFP fusion proteins were treated with DAPI at a final concentration of2 μg/ml for 20 min at 37 °C prior to imaging. Fluorescence from DAPIand GFP was observed and captured from live cells within 30 min ofmounting the samples under the cover slip on a glass slide usingNikon TE 2000-U Nikon TE 2000-U fluorescence microscope.

2.9. ATPase assay

The ATP hydrolysis, catalyzed by PfPrp16he and PfPrp16he+ha

proteins, was assayed by measuring the release of radiolabelledPi from [γ‐32P]ATP (19). The standard reaction mixture (10 μl)contained 20 mM Tris–HCl pH 8.5, 1 mM MgCl2, 30 mM KCl, 4%(w/v) sucrose, 80 μg/ml BSA, 1 mM ATP, 1665 Bq [γ‐32P]ATP(185 Tbq/mmol) and individual recombinant protein. The reac-tion was performed both in the presence and absence of ssRNAfor 30 min at 37 °C and was stopped by chilling to 0 °C. One mi-croliter of the sample was spotted on to a polyethyleneimine cel-lulose thin layer strip (0.6×6 cm) (Sigma) and ascendingchromatography was performed in 0.5 M LiCl and 1 M formicacid (HCOOH) at room temperature for about 15 min. The stripwas dried at room temperature and exposed for autoradiographyto identify the released Pi from ATP. The amount of Pi releasedfrom ATP was determined by excising the Pi and ATP area fromthe plate and determining the counts in a liquid scintillation

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counter. Percentage ATP hydrolysis was calculated by followingformula

% ATPhydrolysis¼ ½spotdensityofreleasedPi=ðspotdensityofunhydrolyzedATP

þ spotdensityofreleasedPiÞ� � 100

2.10. In vitro DNA/RNA binding assay

For the RNA binding assay, different amounts of PfPrphe or PfPrphe+ha

were incubated either with a 33 base 32P-labelled RNA or with a 54base 32P-labelled DNA in binding buffer (50 mMNaCl, 10 mMMgCl2,10 mM HEPES, 0.1 mM EDTA, 1 mM DTT, 1.5% BSA) at 25 °C for30 min. After completion of reaction, reaction mix were run on a10% native PAGE. Gels were subsequently dried and visualized byPhosphorimager (Amersham Biosciences). For equilibrium bindingconstant analysis, the amount of free and bound probe was quantifiedusing ImageQuant 5.2 (Amersham Biosciences). Nonlinear regressionanalysis in saturation bindingmodule of SIGMAPLOTwas used to deter-mine the dissociation constant. In summary, the percent of RNA/DNAbound was plotted against the concentration of protein in each bindingreaction. Dissociation constants, Kd values, were obtained by fitting theexperimental data of at least three replicates frommidpoint of the curveto theHill equation, Y=Bmax*X/(Kd+X),where Y and X are the concen-tration of the protein–RNA/DNA complex and unbound probe respec-tively whereas Bmax is the maximum binding.

2.11. Helicase assay

Nucleic acid unwinding activity of PfPrphe and PfPrphe+ha recom-binant proteins was measured using the standard strand displace-ment assay as described by Pradhan et al. [19]. Briefly, to measurethe DNA unwinding activity, partial DNA duplex was prepared byannealing 54 bases long 5′‐atggcatttatgcttatgcTCGTCCTTGTAGTCTACgacatatgctagcgataa‐3′ with 17 bases long 5′ 32P-labelled 5′‐GTAGACTACAAGGACGA‐3′ DNA oligonucleotides. For RNA unwinding assay,partial RNA duplex was generated by annealing a 33 bases long 5′‐ccaucgauaaAAAAUAUGGAGAGCuucccgaag‐3′ with 14 bases long 5′ 32P-labelled 5′‐GCUCUCCAUAUUUU‐3′ RNA oligonucleotide. A total of10 ng of DNAor RNAoligonucleotidewas labelled at 5′ endwith T4 poly-nucleotide kinase (5 U) and 0.925 MBq [γ-32P]ATP (185 TBq/mmol) in50 mM Tris–HCl pH 7.5, 10 mM MgCl2, 5 mM DTT, 0.1 mM spermidineand 0.1 mM EDTA at 37 °C for 1 h and then incubated at 95 °C for2 min. The labelled DNA or RNA oligonucleotide was annealed to itscomplementary oligonucleotide in 40 mM Tris–HCl pH 7.5, 10 mMMgCl2, 50 mM NaCl and 1 mM DTT. The mixture was heated at 95 °Cfor 2 min and then allowed to cool slowly till it reached the room tem-perature. The substrate was purified by gel filtration through a 2 mlsepharose 4B or blue sepharose CL 6B column. The substrate was elutedin STE buffer (Tris 10 mM, 100 mMNaCl, 1 mM EDTA) and the fractionswere stored at−20 °C.

The helicase assay measures the displacement of 32P labelled oligo-nucleotide from a partially duplex helicase substrate. The reaction wasperformed in a 10 μl reaction mixture consisting of 20 mM Tris–HClpH 8.0, 1 mM ATP, 1 mM MgCl2, 75 mM KCl, 8 mM DTT, 4% (w/v) su-crose, 80 μg/ml BSA, about 1 ng of each substrate (2000–3000 cpm)and PfPrp16he or PfPrp16he+ha pure proteins. The reaction mixtureswere incubated at 37 °C for 1 h and the reactionwas stopped by the ad-dition of 2 μl loading buffer (75 mM EDTA, 2.25% SDS, 37.5% glyceroland 0.3% bromophenol blue). The products were separated by 12% na-tive polyacrylamide gel electrophoresis in 0.5× TBE. The boiled reactionsampleswere kept in boilingwater for 5 min, quenched in ice and load-ed on to the gel after the addition of loading buffer. Gels were fixed afterelectrophoresis in fixing solution (10% methanol, 10% acetic acid) for5 min, transferred to 3 MM Whatman paper, dried in the gel dryer

and autoradiographed. The DNA/RNA unwinding was quantified by ex-cising the bands from the dried gels and counting in Beckman Readysafe liquid scintillation fluid. DNA/RNA Helicase activity was measuredas percentage of unwinding of DNA/RNA duplex by following formulasrespectively.

% DNAunwinding¼ ½spotdensityofsinglestrandDNA=ðspotdensityofdoublestrandDNA

þ spotdensityofsinglestrandDNAÞ� � 100:

% RNAunwinding ¼ ½ðspotdensityofsinglestrandRNAinactualreaction−spotdensityofsinglestrandRNAincontrolreactionÞ=ðspotdensityofdoublestrandRNAþspotdensityofsinglestrandRNAÞ� � 100

2.12. Splicing assay and Northern blot analysis

A 488 bp 3′-end region containing 3rd and 4th exon along with3rd intron of cop-coated vesicle membrane protein p24 precursor(Pf13_0082) was amplified using 82-F and 82-R primers and clonedin to pGEMT easy vector. Orientation of the insert in pGEMT Easywas confirmed by sequencing. The T7 promoter of pGEMT Easy vec-tor is positioned immediately upstream of multiple cloning regions(hence could be used as a transcription template for in vitro tran-scription. Linearized plasmid DNA (1 μg) was used to synthesizeradiolabelled RNA transcript using RiboMax kit (Promega) and 50 μCi of[γ-32P]UTP (3,000 Ci/mmol; Perkin Elmer) following the manufacturer'sinstructions.

The immunodepletion of PfPrp16pwas performed by using the poly-clonal antibodies against the protein raised in rabbit. Briefly, the nuclearlysate was first incubated with anti-PfPrp16p IgG in nuclear lysate ex-traction buffer with varying concentration of sodium chloride (200,400 or 600 mMNaCl) followed by removal of antigen-antibody complexby Protein A-Sepharose beads. Subsequently bound beads were washedthricewith corresponding immunoprecipitation buffers at 4 °C. To checkthe specificity of depletion, respective Protein A-Sepharose beads wereboiled to dissociate the proteins from beads. Proteins were subsequentlyseparated on 8% SDS‐PAGE and silver stained. Depletion of PfPrp16pfrom the nuclear extract was determined by probing the nuclear lysatesfor the presence of protein before and after depletion.

Splicing reaction was carried out as described by Schneider et al.[20]. Briefly, the in vitro transcribed 488 bp PF13_0082 pre-mRNA wasincubated with intact, PfPrp16p immunodepleted or boiled nuclear ex-tracts for 30 min at 25 °C in reactionmix containing 2 mMATP, 2.5 mMMgCl2, 3% PEG 8000, 0.6 M KH2PO4, RNasein, nuclear extract 50% v/vand radiolabelled RNA (40000 cpm). On completion of splicing reac-tions, mixtures were treated with proteinase K (50 mg/ml) and RNAwas extracted with Trizol following the manufacturer's instructions.RNA was fractionated by denaturing PAGE (5%, 7 M urea) and splicingproducts were visualized by autoradiography.

Northern blot analysis of splicing products was performed as de-scribed by Qian et al. [21]. Four complementary DNA oligonucleotideprobes (Supp. Table 2) were synthesized for different regions of splicingsubstrate and 25 pmole of each oligonucleotide was end labelled by T4polynucleotide kinase and [γ-32P]ATP. Splicing products of a cold splicingreaction were separated as described above. Electro-transfer of nucleicacids to nylon chargedmembranes (Immobilon-NY+,Millipore)was car-ried out at 200 mA for 2 h in 0.5× TBE buffer. The cross-linkingwas doneby exposing the membrane under UV rays. Blots were pre-hybridized for3 h at 50 °C in pre-hybridization buffer (6× SSC, 5× Denhardt's solution,0.5% SDS, 1 μg/ml denatured fragmented single‐stranded DNA) and hy-bridized overnight with end-labelled oligonucleotide probes. Membraneswere washed with washing solution (3× SSC, 25 mM Na2HPO4, 5× SDS,10× Denhardt's solution) at 50 °C and finally washed with 1× SDS and

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SSC. Membranes were subsequently visualized by Phosphorimager andwere analyzed using ImageQuant 5.2 (Amersham Biosciences).

2.13. Yeast complementation assay

2.13.1. Constructs for yeast complementation assayFor full length complementation clone PfPrp16 was amplified

using P. falciparum cDNA and PfPrp16-13 and 14 primers. AmplifiedPCR product was digested with BamH1 and Not1 restriction enzymesand cloned in frame in pRS315 yeast expression vector. For construc-tion of chimeric constructs Yeast-Pf173-Prp16 and Yeast-Pf380-Prp16,first 894 nucleotides of yeast Prp16were amplified using yeast cDNAlibrary and yPrp16F (Spe1)–yPrp16R (Sal1) primer pair. Amplifiedfragment was cloned into Topo-TA cloning vector, generatingTopo-Yeast-N-Prp vector. Next, regions of PfPrp16 (517–3456 and1138–3456 nucleotides) were amplified from P. falciparum cDNA li-brary using PfPrp16-15, PfPrp16-14 and PfPrp16-16, PfPrp16-14primers respectively. Amplicons were digested with Sal1 and Not1restriction enzymes and cloned separately into Topo-Yeast-N-Prpvector on corresponding sites. Finally, chimeric constructs were re-stricted with enzymes Spe1 and Not1 and cloned into pRS315 yeastexpression vector.

2.13.2. Genetic crosses and sporulation to create haploids ts prp16-2,leu− spores to be used in complementation studies

The S. cerevisiae prp16-2 mutant haploid (genotype mat-α prp16-2(prp23-1), his3 Δ 200, ura3-52, lys2-801) [22] was crossed to atemperature resistant (PRP16 wild type allele) leu− strain: BJ2168(mat-a, ura 3–52, trp1, leu 2–3, leu 2–112) with the aim of isolatingprp16-2, leu− haploid strain for complementation experiments. Diploidsfrom the above cross were obtained by selection on SD leu− lys−. Fivediploidswere colonypurified and then transferred topre-sporulationme-dium for 16 h followed by plating on minimal sporulation medium (1%potassium acetate, 2% agar) as per standard protocols. From one ofthese sporulating diploid patches random spore populations wereprepared and plated on YPD plates. One hundred sixty‐five putativespore clones were picked on to a master YPD plate and were thenspotted/tested for growth on YPD at 23 °C, 30 °C, 37 °C and SD-leu,SD-his, SD-lys, SD-ura, SD-trp. As controls the parent diploid, thetwo haploid parents (i.e., the original prp16-2 strain) and wild typeBJ2168 were also plated. From the growth pattern on YPD at 37 °C,three temperature‐sensitive leu− haploids were identified. The hap-loid 15 (genotype:mat‐a, prp16-2 ts, leu 2–3, leu2-112, lys2-801, trp1,ura 3–52) was selected for complementation experiments. Beforetransformation with experimental Prp16 expression constructs, this tsleu− strain was transformed with a yeast genomic clone for PRP16 inthe URA3 marked plasmid YCp50. The transformants were selected onSD-ura- and then tested for complementation of the ts phenotype.Strain 15–1 transformed with YCp50PRP16 (PRP23) was temperatureresistant confirming the ts chromosomal allele in this strain as prp16-2.

2.13.3. Transformation of Prp16 expression constructs into a prp16-2 tsstrain and complementation analysis

The yeast haploid strain 15–1 was transformed with the plasmids:Yeast-Pf173-Prp16 in pRS315-GalUAS, Yeast-Pf380-Prp16 in pRS315-GalUAS and PfPrp16 in pRS315-GalUAS, the vector pRS315-GalUASand the plasmid ScPRP16 in YCp50. In each case ~4 μg of DNA wastransformed into the 15–1 strain and transformants selected onSD-leu (for pRS315 plasmids) or SD-ura (for Yp50 plasmid). Singlecolonies were picked (two for each transformant), grown and used incomplementation studies. The transformants and control untransformedprp16-2 strains were grown in broth culture at 23 °C with appropriateselection for the transformed plasmid for 12–14 h to obtain log phasecells of O.D. ~1.0. The control untransformed prp16-2 strain was grownin richmedia at 23 °C. The cells fromall cultureswere then appropriatelydiluted to obtain an O.D. of ~0.5 in 100 μl of sterile MQ water and then

this was serially diluted to obtain 10−1, 10−2, 10−3 and 10−4 dilutions.Five microliters of these dilutions was spotted on YPD and YPGal Raffplates as required. These plates were air-dried and incubated at differenttemperatures (23, 30 and 34 °C) for 48–60 h and growth profile of thestrains was monitored.

2.14. Molecular modeling of PfPrp16p

X-ray crystal structure of a closely related yeast Prp43p splicinghelicase, having similar domain architecture, was available [23].Three dimensional model of PfPrp16p was built on Prp43p by sub-mitting the protein sequence at I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) [24]. Briefly, Prp43p structure wasretrieved from the PDB library. In the second step, the continuousfragment excised from the PDB template was used to model the ho-mologous region of PfPrp16p by replica-exchange Monte Carlo sim-ulations. Structure of unaligned regions was generated by ab initiomodelling. Subsequently, the fragment assembly simulation wasperformed to remove the steric hindrance as well as to refine theglobal topology of the cluster centroids.

3. Results

3.1. Identification of P. falciparum homologues of yeast DExD/DExH-boxcontaining splicing factors

Canonical intron features i.e. GU at donor site and AG at acceptorsite have been successfully utilized to annotate the exon–intron bound-aries in Plasmodium spp. and these features suggest the presence of aconserved mechanism of intron splicing in malaria parasite [25].In silico search for DExD/DExH-box splicing factors in P. falciparumgenome was performed using yeast splicing helicase sequences asthe query. This resulted in the identification of PFL1525c,PFE0430w, MAL13P1.322, PF10_0294, PFE0925c, PFI0860c andPFD1060w as putative homologues of yeast Prp2, Prp5, Prp16,Prp28, Prp43 and PfBrr2 respectively with translated protein se-quence identity ranging from 19% to 49% and similarity varyingfrom 32% to 63% (Supp. Fig. S1A). Among the two candidate genesMAL13P1.322 and PF10_0294, identified as likely homologues foryeast and human Prp16, we predicted MAL13P1.322 as the best candi-date for P. falciparum Prp16 based on the domain organization and %identity (Supp. Fig. S1B). The other candidate gene, PF10_0294contained an extra S1 domain at the N-terminus, a property of Prp22phelicases, suggesting that PF10_0294 is most likely a P. falciparum ho-mologue of yeast Prp22 (Fig. 1A). A global alignment of PF10_0294 pro-tein with other Prp22p homologues showed a higher homologybetween them than the yeast Prp16p, ascertaining it as a putativePfPrp22p.

A search for P. falciparum homologue of Brr2p also yielded twocandidates, PFD1060w and PF14_0370. Both these candidates pos-sess similar domain architecture as that of yeast Brr2p except thatthe second helicase domain of candidate PF14_0370 also containsthe Helicase-C region as seen in case of human Brr2p (Supp. Fig.S1C). To the best of our knowledge, two helicase domains are pres-ent only in Brr2p and a highly related Slh1 protein [26]. Based onthese observations, we performed the global as well as domain-wise alignment to identify the P. falciparum Brr2p candidate gene.The candidate PFD1060w showed better homology with Brr2 pro-teins, whereas PF14_0370 has slightly higher homology with Slh1class of proteins (Supp. Fig. S1D, panel i). Domain-wise analysis ofPFD1060w with Brr2p and Slh1p domains further indicated thatPFD1060w is the likely P. falciparum homologue of Brr2 (Supp. Fig.S1D, panel ii). The occurrence of these genes in P. falciparum hasbeen predicted in a recent review by Tuteja [27].

Fig. 1. Expression and purification of PfPrp16p protein domains. (A) Domain organization of P. falciparum DExD/DExH-box splicing factors. (B) Schematic representation of the fulllength PfPrp16p. Numbers above schematic refer to the position of amino acid residues defining various domains. The three bacterially expressed recombinant protein domains ofPfPrp16p, PfPrp16ph, PfPrp16he and PfPrp16he+ha, are shown by the horizontal lines. (C) SDS-PAGE analysis of the purified recombinant PfPrp16p domains, PfPrp16ph, PfPrp16he andPfPrp16he+ha.

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3.2. Sequence features, expression and characterization of PfPrp16p

The ClustalW alignment of putative P. falciparum Prp16p sequencewith other homologues from S. cerevisiae (NP_013012), S. pombe

Fig. 2. Expression of the PfPrp16p during asexual blood stages of P. falciparum. (A) Semi-quashows expression levels in ring (R), trophozoite (T) and schizont (S) developmental stagesparasite lysates detecting the PfPrp16 protein levels across the erythrocytic developmentsub-cellular fractions, cytoplasmic (C) and nuclear (N), in ring (R), trophozoite (T) and schthe first panel Pf-histone h2A and Pf-actin were probed to show equal protein loading aassay based localization of the PfPrp16p. Anti-PfPrp16p sera (1:200 dilution) were used to lostages. DAPI and anti-Pf‐Tudor-SN (PfTSN) antibody were used to localize the nucleus.

(CAB52799), human (AAC27431),Arabidopsis (Q38953), Caenorhabditis(P34498) and Chlamydomonas (AF305070) showed highly conservedcentral helicase and C-terminal domains while low level conservationwas observed in the N-terminal region (Supp. Fig. S2A). Using a set of

ntitative RT-PCR analysis of PfPrp16 transcript levels, determined using specific primers,. α-Tubulin transcript was detected as the loading control. (B) Western blot analysis ofal stages. (C) Western blot analysis to determine distribution of the PfPrp16p in twoizont (S) developmental stages. Sub-cellular distribution of the PfPrp16p is shown innd to detect any nucleo-cytoplasmic cross‐contamination. (D) Immunofluorescencecalized the protein using thin blood smears of P. falciparum ring, trophozoite and schizont

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gene-specific primers, the Plasmodium Prp16 (PfPrp16) cDNA wasPCR amplified, cloned and sequenced. PfPrp16 gene encodes a 1151amino acid protein consisting of a N-terminal non-conserved region,followed by a conserved helicase domain, helicase-associated region2 (HA2) and a C-terminal domain of unknown function (DUF1605)(Fig. 1B). The sequence analysis confirmed the presence of all theconserved motifs (I, Ia, Ib, Ic, II, III, IV, V and VI at amino acid posi-tions, 502–509, 530–537, 556–564, 576–580, 599–602, 631–633,698–701, 802–811 and 849–860) typical of the DExH-box helicasefamily. Orthologs having similar domain organization were alsoidentified in other Plasmodium species (Supp. Fig. S2B).

To characterize PfPrp16p biochemically, we cloned full lengthPfPrp16p in different E. coli expression vectors, such as pQE-30, pET-28,pMAL-p2X and pGEX-4T-3. However, all our attempts to express theprotein in a reasonable amount were unsuccessful. Subsequently, thegene was divided into three distinct biochemical domains: PfPrp16ph(prehelicase region, 1–465 aa), PfPrp16he (helicase domain, 466–866 aa) and PfPrp16he+ha (helicase and helicase‐associated regionstogether, 466–1010 aa). These domains were expressed by cloningin E. coli pQE-30 and proteins were expressed upon induction withIPTG (Fig. 1B and C). PfPrp16ph recombinant protein compartmental-ized in inclusion bodies, whereas PfPrp16he and PfPrp16he+ha re-combinant proteins were efficiently produced in soluble form.PfPrp16ph recombinant protein was purified on a Ni2+-NTA affinitycolumn under denaturing conditions and was refolded by gradual

Fig. 3. N-terminal domain of the PfPrp16p targets the protein to the nucleus. (A) Schematicsequence, “KRRK,” is shown as a black box at 41st–44th amino acid position. (B and C) Immuand (C) schizont stages respectively. Anti-GFP (red, 1:200 dilution) and anti-PfTSN (grPfPrp16.1-40-GFP and PfPrp16.81-last-GFP chimeric proteins do not localize with the nucleaPfPrp16.1-80-GFP was trafficked to the nucleus in trophozoite and schizont stages.

removal of urea up till urea concentration reached of 1 M. Any fur-ther reduction of urea in the purified protein preparation resultedin precipitation. PfPrp16he and PfPrp16he+ha recombinant proteinswere purified to homogeneity using Ni2+-NTA affinity columns in thenative conditions (Materials and methods). Purified recombinant pro-teins PfPrp16ph, PfPrp16he and PfPrp16he+ha migrated on SDS-PAGEgels with a molecular weight of ~55, ~45 and ~63 kDa respectively inaccordance with the expected theoretical molecular mass (Fig. 1C).

3.3. Stage specific expression and localization of PfPrp16p

Expression and the relative abundance of PfPrp16 transcript at dif-ferent asexual blood stages—ring, trophozoite and schizont—were an-alyzed by semi-quantitative RT-PCR. The RT-PCR analysis showed thatPfPrp16 is transcribed in all the three asexual blood stages, though theexpression level was comparatively reduced in the trophozoite stage(Fig. 2A). To further confirm these expression data, western blot anal-ysis was performed with parasite lysates prepared from three asexualblood stages and the protein was detected using affinity purifiedanti-PfPrp16p antibodies. As shown in Fig. 2B, PfPrp16p protein wasdetected in all the three asexual blood stages of the parasite.

We next analysed the sub-cellular distribution of PfPrp16p by im-munoblotting and fluorescence microscopy based co-localization stud-ies. In the ring stage parasites, the protein was mainly seen in thenucleus as evident by western blot analysis of nuclear and cytoplasmic

representation of the PfPrp16-GFP chimeric constructs. Predicted nuclear localizationnofluorescence assay based localization of the chimeric GFP proteins at (B) trophozoiteeen, 1:100 dilution) antibodies were used on thin blood smears of parasite lines.r marker PfTSN protein in either of the developmental stages, whereas chimeric protein

Fig. 4. Biochemical characterization of the PfPrp16he and PfPrp16he+ha recombinant proteins. (A) ATPase assay for PfPrp16he and PfPrp16he+ha was carried out at different proteinconcentrations. The released radiolabelled Pi from [γ-32P]ATP was separated on PEI-TLC plates. Percentage ATP hydrolysis was calculated as described in the Materials and methodssection. Protein concentration used and the ATPase activity observed are mentioned below the panel. Lane 1: Control reaction in absence of any added protein, lane 2: reactioncarried out in the presence of BSA, lane 3: reaction in the presence of corresponding heat denatured proteins. (B) PfPrp16he and PfPrp16he+ha protein ATPase activity in the presenceof single-strand RNA. Lanes 1–3 are the controls as described above except the RNA was also added in each reaction. (C) Interaction of PfPrp16he and PfPrp16he+ha with the RNA asshown by the EMSA analysis. Radiogram of the gels for RNA binding activity of PfPrp16he and PfPrp16he+ha respectively. (D) Radiogram of the gels for DNA binding activity ofPfPrp16he and PfPrp16he+ha respectively. (E) Plot of the fraction RNA bound as a function of the respective protein concentration. Quantitation of the data from RNA EMSA exper-iments, as determined for each protein, was done and data was fit to Hill equation for the calculation of dissociation constant as described in the Materials and methods section. Datapoints reported are the average±SD for three independent experiments for RNA binding analysis. (F) Quantitative analysis and dissociation constant derived from four indepen-dent DNA EMSA experiments for each protein as described in E.

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fractions of parasitic extracts (Fig. 2C). These were consistent with itsco-localization with Pf-Tudor-SN (PfTSN), a nuclear resident protein[28] (Fig. 2D). However, at the trophozoite and schizont stages of para-site development, the protein was detected in the nucleus as well as inthe cytoplasm indicating nucleo-cytoplasmic shuttling of PfPrp16p(Fig. 2C and D). Pf‐histone h2A (nuclearmarker) and Pf‐actin (cytoplas-mic marker) were probed to confirm equal protein loading and to ex-amine any nucleo-cytoplasmic cross-contamination during extractions(Fig. 2C).

3.4. N-terminal sequence (1–80 amino acids) of PfPrp16p is required fornuclear targeting

Since PfPrp16p is mainly nuclear localized, we next dissected theminimal amino acid sequences required for PfPrp16p targeting tothe nucleus. Three transgenic parasite lines that contained GFP

reporter fused to the C-terminal of PfPrp16 sequence were generated(Fig. 3A). Parasite lines expressing chimeric proteins were obtainedbetween 50 to 60 days in the presence of 2.5 μΜ antifolate drug,WR99210 and expression of the chimeric GFP proteins was confirmedby western blot analysis (Supp. Fig. S3). The distribution of GFP inthese transgenic lines was studied in fixed cultures using anti-GFP an-tibodies followed by fluorescence microscopy as well as by live cellimaging of GFP (Fig. 3B and Supp. Fig. S3). PfPrp16.1-40-GFP chimericprotein that contained the first 40 amino acids of PfPrp16p was nottargeted to the nucleus; its distribution was mainly cytoplasmic(Fig. 3B and C and Supp. Fig. S3A, panel i and ii). In comparison,PfPrp16.1-80-GFP fusion protein that contained the first 80 aminoacids of PfPrp16p was correctly targeted to the nucleus as evidentfrom its localization with DAPI stain and co-localization with PfTSNprotein (Fig. 3B and C and Supp. Fig. S3B, panel i and ii). PfPrp16.81-last-GFP protein that expressed 81–1151 amino acids of PfPrp16p fused

Fig. 5. Nucleic acid unwinding activities of the PfPrp16he and PfPrp16he+ha recombinant proteins. (A and B) DNA and RNA duplex unwinding activities of the PfPrp16he. (C and D)DNA and RNA unwinding activities of the PfPrp16he+ha protein. Protein concentration in each reaction is provided below the panel. Lane labelled as control-1 is a reaction withoutenzyme, lane labelled as Control-2 is the reaction with BSA alone, and lane labelled as “boiled” is the reaction in which substrate was heat denatured. In RNA unwinding assays,complete removal of single‐strand labelled RNA from annealed RNA duplex preparation could not be achieved. Nonetheless the unwinding activity is readily observed and thiswas quantified over and above the background level of ssRNA present in the input sample (B, control-1 and D, control-2). Actual RNA unwinding catalyzed by the recombinantproteins was quantified by subtracting the band density for single‐stranded oligonucleotide in the control reaction from the band density for single‐strand oligonucleotide fromeach of the enzyme catalyzed unwinding reactions (B: lanes 2–4, D: lanes 3–8). The helicase activities were measured as percentage of unwinding of respective duplex by theformula described in Materials and methods section.

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to GFPwas not nuclear andwasmainly localized in the cytoplasm (Fig. 3Band C and Supp. Fig. S3C, panel i and ii). Sequence analysis of PfPrp16pindicated a putative nuclear localization sequence “KRRK” at amino acid

Fig. 6. Homology modelling of full length PfPrp16p by I-TASSER module using yeast Prp43p adomain (NTD,magenta), DEAHdomain (yellow), Helicase_C region (C-terminal region of helicaprotein and helicasemotifs I to VI are indicated. (C) PfPrp16he+ha recombinant protein. Helicasemodel for PfPrp16he+ha.. (E) Interaction between Helicase and HA2 domains shown in the surfT-633 in motif III (SAT) of helicase domain and that of I-957 in HA2 domain with H-602 in mo

positions 41–44. Together these results indicate that the N-terminal1–80 amino acids of PfPrp16p possess a classical nuclear localizationsignal sequence and target the protein to the parasite nucleus.

s a model protein. Modelled structure of (A) the PfPrp16p full length protein, N-terminalse domain, green), HA2 domain (sky blue), DUF1605 (orange). (B) PfPrp16he recombinantmotifs are depicted as in (B). HA2 region is shown in sky blue. (D) Surface representation

ace representation model. (F) Molecular interactions between K-954 in HA2 domain withtif II of helicase domain.

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3.5. Functional characterization of recombinant PfPrp16he and PfPrp16he+ha

proteins

3.5.1. PfPrp16 possesses RNA‐dependent ATPase and nucleic acid bindingactivities

To examine whether PfPrp16p possesses ATP hydrolytic activity,two bacterially expressed recombinant protein fragments of PfPrp16p,PfPrp16he and PfPrp16he+ha, were biochemically characterized for ATPaseactivity. ATP hydrolysis activity of PfPrp16he proteinwasmeasured over awide range of protein concentrations and a dose‐dependent increase inhydrolysis was observed (Fig. 4A). Comparatively, PfPrp16he+ha proteinappeared to be extremely poor for ATP hydrolysis (Fig. 4A) and the ob-served minimal ATPase activity of PfPrp16he protein (1% at 0.2 nM) wasequivalent to that of observedmaximum activity of PfPrp16he+ha protein(1%, 10 nM). Bovine serum albumin (BSA) alone did not cause release ofradioactive phosphate (Fig. 4A). We next assessed the ATP hydrolytic po-tential of each of these proteins, PfPrp16he and PfPrp16he+ha, in the pres-ence of single‐stranded RNA. Both recombinant proteins, PfPrp16he andPfPrp16he+ha, showed significant increase in ATP hydrolysis in the pres-ence of RNA indicating that PfPrp16p possesses an RNA-dependentATPase activity (Fig. 4B).

Subsequently, we assessed the DNA and RNA binding activities of re-combinant PfPrp16he and PfPrp16he+ha proteins in gel retardation assayusing 32P-labelled RNA or single‐stranded labelled DNA. Both PfPrp16heand PfPrp16he+ha proteins were able to bind labelled single-strandedRNA (Fig. 4C). RNA binding increased with increasing protein concentra-tions and was lost on denaturation (boiling) of these proteins. Compara-tively, PfPrp16he protein showed higher affinity binding to single‐strandRNA (Kd=1.21±0.19 nM) than that of PfPrp16he+ha protein (Kd=2.16±0.34 nM) (Fig. 4E). Similarly, both the recombinant proteins

Fig. 7. PfPrp16p acts at the second catalytic step of splicing (A) P. falciparum PF13_0082 gconnected with the introns. The numbers indicate the boundaries of the exons. Region of primassay using P. falciparum nuclear lysates. Primary mini-pre-mRNA transcript was used as thwith boiled nuclear extract and lane NL, labelled transcript incubated with nuclear extractthe right. (C) Target region of northern probes in mini-pre-mRNA transcript is depicted bmini-gene and not at PF13_0082. The curved arrow suggests the region of lariat formationproducts. Splicing reactions with heat inactivated nuclear extract (lane 2), nuclear extract ((lane 5). Immunodepletion of the PfPrp16p (lane 4) arrests the second step of the splicing asbands was confirmed by comparing the hybridization pattern with various probes. All the fmRNA, whereas probe-E1 (exon-1) did not recognize the lariat–exon2.

showed a dose-dependent increase in DNA binding (Fig. 4D) andPfPrp16he displayed higher affinity (Kd=2.03±0.26 nM) than that ofPfPrp16he+ha protein (Kd=3.41±0.47 nM) (Fig. 4F). Taken together,these results suggest that HA2 domain negatively influences the ATPaseas well as nucleic acid binding activities of the PfPrp16p helicase domain.

3.5.2. Nucleic acid unwinding activities of PfPrp16he and PfPrp16he+ha

proteinsNucleic acid unwinding activities were measured for both PfPrp16he

and PfPrp16he+ha proteins by a strand displacement assay using partialDNA or RNA duplexes. As shown in Fig. 5A and B, PfPrp16he could effi-ciently unwind these DNA as well as RNA duplexes in a concentrationdependent manner. PfPrp16he+ha also exhibited both DNA and RNAhelicase activities however this was considerably lower level of activities(Fig. 5C and D). In the case of PfPrp16he, DNA unwinding activity wasobserved even in the presence of 0.5 nM protein (8%) (Fig. 5A), whilePfPrp16he+ha protein showed only 5% activity even at 4 nM concentra-tion (Fig. 5C). Likewise, considerable RNA unwinding activity (25%)was observed with 2 nM of PfPrp16he protein (Fig. 5B), while only 5%RNA helicase activity was detected with 2 nM PfPrp16he+ha protein(Fig. 5D). During RNA unwinding analysis, we also observed slight un-winding of 14 bp RNA duplex in the control reaction (Fig. 5D, lane 2).Nonetheless, the unwinding activity could easily be observed and quan-tified over and above the background level of ssRNA present in the inputsample. To confirm if the partial unwinding of the duplex RNA in theassay was due to lower stability of duplex, we performed the RNA un-winding experiment with a 19mer RNA duplex. With this longer RNAduplex, no unwinding was observed in the control reactions (Supp. Fig.S4, A and B). Although on comparing PfPrp16he activities on these twotemplates it was seen that PfPrp16he showed low unwinding activities

ene organization. All four exons of the gene are depicted by the black boxes and areary mini-pre-mRNA transcript is shown by the converging arrows. (B) In vitro splicinge substrate for splicing. Lane 1, radiolabelled pre-mRNA; lane 2, pre-mRNA incubated. Schematic representation of the splicing intermediates and products are indicated toy double headed arrow. Numbers represents the nucleotide position with respect to. (D) In vitro splicing assay for the role of PfPrp16p and identities of different splicinglane 3), PfPrp16 immunodepleted nuclear extract (lane 4) and mock‐depleted extractlariat–exon intermediate accumulate and spliced mRNA is not detected. Identity of the

our probes identified the input pre-mRNA. The intron probe failed to recognise mature

Fig. 8. In vivo assays for complementation of temperature-sensitive yeast prp16mutant. (A) Schematic representation of the proteins expressed from Gal4 UAS regulatory elements.represents the sequence derived from yeast Prp16 N-terminal region, represents the sequence derived from PfPrp16 N-terminal region, and represents the func-

tional domains (Helicase+HA2+DUF1605) of PfPrp16. Numbers above and below the construct represent the amino acid positions in yeast Prp16 and PfPrp16 respectively. (B andC) Dilution spotting of prp16-2 mutant and various transformants. Growth on YPD media and in Galactose Raffinose media is shown after incubation at different temperatures for24 h (B) and 48 h (C). The untransformed wild type strain and the mutant transformed with empty vector were also plated as controls (spotting details are mentioned in the fig-ure). (D) Western blot analysis showing the expression of PfPrp16p and its fusion derivatives in prp16 mutant cells. Yeast-Pf380-Prp16 chimeric protein levels in uninduced andinduced growth conditions. (E) Yeast-Pf380-Prp16 chimeric protein (lane 1) and full length PfPrp16 (lane 2). (F) Total protein content of each yeast strain at the time of spotting(0 h).

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of recombinant proteins with 19mer duplex, yet notably the activityobserved for PfPrp16he protein was consistently higher than that ofPfPrp16he+ha protein. The reduction in the unwinding activity withthe 19mer duplex may be due to the fact that the processivity ofRNA helicases is inversely proportional to the length of the duplexand DExD/DExH-box proteins, alone, are inefficient in unwinding oflong duplexes with two or more helical turns [29]. Together theseresults suggested that the HA2 domain is an inhibitory/regulatorydomain for PfPrp16p helicase activity.

3.6. Molecular modelling of PfPrp16 and its domains

Based on the crystal structure of yeast DExD/DExH-box proteinPrp43p, we generated a structure for PfPrp16p by homology model-ling using I-TASSER server (Fig. 6). The modelled PfPrp16p structureshowed that the N-terminal non-conserved region (NTD) consists ofeleven α helices and three anti- parallel β sheets (Fig. 6A). ThePfPrp16p helicase domain has a dumbbell shape structure in whichamino- and carboxy-terminal domains are connected by a linkerpeptide which is similar to that of the helicase domain organisationof the eukaryotic elongation factor eIF4a [30]. The N-terminal ofPfPrp16p helicase domain consists of a seven-strand parallel βsheet sandwiched between α helices and motifs I, Ia, Ib, Ic, II and IIIcontained in this domain. The C-terminal segment of the helicase re-gion consists of a five-strand parallel β sheet sandwiched between α

helices and contains motifs IV, V and VI (Fig. 6B). Helicase-associateddomain (HA2) of PfPrp16p has an ordered structure consisting ofthree α helices (Fig. 6C, sky blue). The PfPrp16p model structuresuggested that I-957 of HA2 domain interacts with H-602 of motifII (DEAH) and K-954 of HA2 region directly interacts with T-633 ofmotif III (SAT) of domain (Fig. 6D–F).

3.7. Role of PfPrp16p in the second catalytic step of splicing

We performed a parasite specific splicing assay using an in vitro la-belledmini pre-mRNA transcript, encompassing exon-3 to exon-4 regionof the P. falciparum gene “cop-coated vesiclemembrane protein p24 pre-cursor” (PF13_0082) (Fig. 7A). The labelled pre-mRNA was incubatedwith the nuclear extract prepared from asynchronous parasite culturesand the reaction productswere separated on 5% urea PAGE. The productsof these splicing reactions were detected by phosphorimager analysis(Fig. 7B). Identity of each band was confirmed by northern blot analysisof these RNA species obtained from parallel reactions performed withcold/unlabeled pre-mRNA in splicing reactions. For these northernblots four probes were designed representing exon-1, exon-2, intronand intron/exon2 junction of mini-pre-mRNA transcript (Fig. 7C, Supp.Table 2). As expected, all the probes recognised the input pre-mRNAtranscript (Fig. 7D, lane 1 and corresponding band in lane 2 of eachblot). The band above the input pre-RNA was recognised by intronprobe, intron–exon-2 and exon-2 probes, whereas exon-1 probe failed

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to hybridizewith this RNA species indicating it to be the lariat–exon2 in-termediate. The observed band below input pre-mRNAwas ascertained,to be the mature mRNA as it hybridized only with exonic probes, whereas intron and intron-exon2 probes did not hybridize to this RNA species.Exon-1 and intron probes also hybridized to a band each correspondingto exon1 and intron lariat, migrating ahead of maturemRNA, though thesignal intensitieswere very low. In fact in the standard splicing reactions,these signals are detected only after longer exposures (Supp. Fig. S5Dand E). To determine the role of PfPrp16p in splicing, protein wasimmunodepleted, using purified anti-PfPrp16p antibodies, from theparasite nuclear extracts at different salt concentrations. Specificimmunodepletion of the PfPrp16p (>90%) protein was obtainedfrom these nuclear extract at 600 mM NaCl (Supp. Fig. S5A and B).The PfPrp16p depleted nuclear extract along with mock-depletedextract was assessed for their in vitro splicing activity. Pre-mRNAtranscript was efficiently spliced in the control extracts and inmock-depleted nuclear lysate, whereas accumulation of lariat–exonsplicing intermediatewas observed in PfPrp16p depleted extract.More-over, mature mRNA and excised lariat–intron were not detected inPfPrp16p depleted extract indicating that PfPrp16p is required for effi-cient and complete splicing of the primary transcript to generate ma-ture mRNA (Fig. 7D and Supp. Fig. S5C and D).

3.8. Complementation of a yeast prp16 mutant by P. falciparum Prp16homologue

To determinewhether the PfPrp16 cDNA is a functional homologue ofyeast Prp16, we studied the ability of PfPrp16 to complement growth de-fects of the yeast prp16-2 temperature-sensitivemutant strain. Altogeth-er three expression constructs were designed to express a wild typePfPrp16 and two fusion proteins containing yeast Prp16 and P. falciparumPrp16 sequences. The first chimeric protein yeast-Pf380-Prp16 was gen-erated by creating a recombinant construct to express translation fusionof 297 amino acid from the N-terminal domain of yeast Prp16 withamino acid residues 380–1151 of PfPrp16. The resultant chimera had anN-terminal domain of 385 amino acid followed by the functional do-mains. The other chimeric protein expression construct yeast-Pf173-Prp16 had the identical N-terminal region of yeast Prp16 fused to173–1151 amino acid coding region of PfPrp16 resulting in a longer,592 amino acid, N-terminal domain (Fig. 8A). These recombinant con-structs were fully sequenced to ascertain the fusion junctions and thecontinuity of the reading frames. All the three expression constructswere tested alongwith yeast PRP16 gene, as a control, for complementa-tion of the growth of prp16-2mutant strain after 24 h (Fig. 8B) and 48 h(Fig. 8C) of incubation at different temperatures. As expected, the wildtype strain (Fig. 8B and C, lane 1) as well as yeast mutant strainstransformed with yeast PRP16 containing plasmid (lane 6) grew well.In contrast, the transformants with the empty yeast expression vector(lane 2) or the expression vector with the full length PfPrp16 (lane 3)did not rescue the prp16-2 ts phenotype even after 60 h of growth onplates. Significantly yeast-Pf380-Prp16 did rescue the growth defect ofprp16-2 mutant (lane 5). Interestingly, the other chimeric proteinyeast-Pf173-Prp16 (lane 4) failed to rescue the mutant even thoughboth the chimeric proteins are expressed as noted by western blot anal-ysis (Fig. 8D and E). Taken together, results of these complementationassays show one of the two chimeric proteins, yeast-Pf380-Prp16, com-plements the yeast prp16-2 mutant and thus indicate that PfPrp16 is afunctional homologue of yeast Prp16.

4. Discussion

Introns have been predicted in ~51% of Plasmodium genes; thus,the abundance of intron containing genes is roughly similar to othereukaryotic models like Schizosaccharomyces pombe and Dictyosteliumdiscoideum [25]. In the malaria parasite, gene regulatory mechanismsare not well understood and introns splicing are believed to be a

major regulatory event that can modulate the generation of distinctproteins via alternate splicing of pre-mRNA transcripts [31,32]. How-ever, to date little information is available about the parasite splicingfactors and splicing mechanism. Removal of introns occurs in thespliceosome which undergo multiple sequential conformational changesduring assembly and splicing catalysis [2,3]. DExD/DExH-box family ofproteins are an important group of splicing factors which govern confor-mational changes of the spliceosome. In the present study, seven such fac-tors, PfPrp2p, PfPrp5p, PfPrp16p, PfPrp22p, PfPrp28p, PfPrp43p andPfBrr2p,were identified in Plasmodium genomeusinghomology searches.The deduced amino acid sequences of splicing related DExD/DExH-boxproteins showed that these proteins possess the typical signature motifsin helicase domain and have the conserved domain organization seen intheir yeast and human homologues.

Expression and localization studies of PfPrp16p showed that theprotein is expressed in all three intra-erythrocytic stages and ispre-dominantly localized in the nucleus as seen in the case of yeastand human Prp16p homologues [33,34]. However, at the schizontstage, nucleo-cytoplasmic distribution of the PfPrp16p was observed.Similar stage specific distribution has been recently reported for someof the P. falciparum splicing factors such as SR protein (PfSR1), SR pro-tein kinase 1 (PfSRPK1) and splicing related CDK-like kinase, PfCLK2[35,36]. It is possible that nucleo-cytoplasmic shuttling is a mean ofregulating the concentration of protein in nucleus as has beenreported for human Slu7, hnRNP1 A1 and polypyrimidine tract bind-ing splicing factors [37–39]. Moreover, it is also possible thatPfPrp16p may have additional roles in parasite development. In fact,two of the Prp16 homologues, Mut-6 of Chlamydomonas reinharditiand MOG-1 of Caenorhabditis elegans, have been shown to functionin trans-gene silencing and sex differentiation respectively [17,40].GFP reporter based assays were performed to decipher the nucleartargeting region of PfPrp16p. Imaging of different transgenic linessuggests that the localization signal resides between 41st to 80thamino acid of PfPrp16p. A putative monopartite nuclear localizationsignal sequence “KRRK” was identified by bioinformatic searches at41st to 44th amino acid positions.

Although a large number of Plasmodium genes undergo splicing, invitro assays have not been used to study splicing reactions in detail.We used our previously described assay [36] and analysed the splicingreaction progress in the presence and after depletion of PfPrp16. Plas-modium nuclear extract efficiently spliced the input mini-pre-mRNAtranscript, while lariat–exon2 and lack of spliced mRNAwas seen in re-actionswhere Prp16pwas depleted from the extract. The identity of thesplicing intermediates was confirmed by northern blotting. These ex-periments thus elucidate a role of PfPrp16 in the second step of splicing.This role of PfPrp16p was further confirmed by a complementationassay using temperature‐sensitive yeast prp16mutant strain. A chime-ric construct, yeast-Pf380-Prp16, rescued the temperature inactivationtriggered growth defect of the ts yeast mutant, whereas full lengthPfPrp16was unable to complement the yeast mutant. These results fur-ther strengthen the inference that assembly of the heterologousPfPrp16 protein into yeast spliceosomes requires the yeast specificN-terminal domain of Prp16p [41]. Interestingly another chimeric con-struct yeast-Pf173-Prp16 could not rescue ts prp16-2 mutant in spite ofthe fusion having the identical yeast specific N-terminal region and de-spite the comparable expression of these fusion proteins in vivo. Thedifferential rescue by the chimeric constructsmay be due to the similar-ity in the length of the N-terminal domain of yeast Prp16 (353 aminoacids) and the N-terminal domain used in the yeast-Pf380-Prp16 chi-meric protein (385 amino acid). The extended N-terminal domain inthe fusion yeast-Pf173-Prp16 (592 amino acids) may have failed tomaintain the relative spatial distribution between its helicase (function-al) core and spliceosome. Another possibility may be the inability ofyeast-Pf173-Prp16 chimeric protein to adopt the right conformation.Taken together, our results unequivocally indicate the role of PfPrp16pin second catalytic step of splicing.

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An important finding arising from the present study was consider-ably lower ATP hydrolysis activity, lower nucleic acid binding capacityas well as reduced helicase activity of the PfPrp16he+ha domain in com-parison to the PfPrp16he protein domain. These findings of ours suggestthat the helicase‐associated regionmay inhibit/regulate these importantbiochemical properties of PfPrp16p. The “intrinsic” high ATPase activityof PfPrp16he appears to be governed by high rate of ATP hydrolysiswhereas higher “RNA stimulated” ATPase activity could be due to thecombination of high intrinsic ATPase activity as well as higher RNA af-finity of PfPrp16he than that of PfPrp16he+ha. The role of HA2 region ininhibition/regulation of PfPrp16 biochemical activities is furthersupported by our homology modelling analysis. In DExD/DExH-boxhelicases, motif II along with motif I, IV and VI, creates the ATPbinding/hydrolysis pocket of DExD/DExH-box helicases, whereasmotif III is involved in coupling of the ATPase and nucleic acid un-winding activities [29]. Observed interaction of HA2 domain withmotif II and III of helicase domain may curtail the functional associ-ation between different PfPrp16p helicase motifs and ATP/RNA andthus influence the ATPase and helicase activities of the protein.

Though the role of Prp16 is well established in second catalyticstep of splicing, molecular mechanisms by which it acts are notwell understood. Nonetheless, ATPase activity of Prp16 has beenlinked to the commitment of the splicing intermediate products gen-erated from the first trans-esterification reaction to enter either pro-ductive pathway to generate mature mRNA or to a discard pathwaythrough which aberrant splicing intermediates are degraded [2,3].Prp16 mutant alleles, with decreased ATP hydrolysis activity, havebeen shown to stabilize the aberrant intermediates at second cata-lytic step and facilitate the generation of mature mRNAs thus rescu-ing them by entering in to discard pathway [42]. It is possible thatwild type Prp16 protein, in itself, also has a similar ability of suppres-sion of ATPase activity to a critical level via its helicase-associateddomain, HA2, which can maintain the balance between stabilityand fidelity at second catalytic step. Implication of such a balancecan be critical in many organisms where branch site consensus ele-ment are less conserved and where such authentic splicing sitesmay be treated as mutated/aberrant transcript if Prp16 functionsare not modulated. Regulated Prp16 activity may also be relevantin cases where the conservation of alternate splice sites issuboptimal to splicing consensus branch sites [43]. It will be worthto explore the molecular mechanism of PfPrp16 action and to under-stand modulatory effects of the HA2 domain on Prp16 in vivoactivities.

4.1. Conclusions

In summary, we functionally characterize one of the members ofDExD/DExH-box family of splicing factors in Plasmodium genome,Prp16 homologue of P. falciparum. Biochemical activities, in vitro splic-ing and in vivo yeast complementation assays demonstrate that thisprotein is a homologue of its human and yeast counterparts. Our workalso shows that helicase-associated region “HA2” can modulate thebiochemical activities of PfPrp16. Our findings demonstrate that thefull length PfPrp16 cannot fully substitute for yeast Prp16 function,suggesting subtle organism specific differences amongst homologuesof Prp16p [41]. Interaction studies in yeast have shown that Prp16passociates with the late acting protein factors within the spliceosome[44]. Moreover, recent advances also suggest that yeast homologuecan be associated with first catalytic step of splicing [13,45]. It willbe interesting to identify the interacting factors of P. falciparumPrp16p and study their associations in the spliceosome assembly.Any differences in their associations can then be exploited for thedevelopment of Plasmodium specific inhibitors.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.bbagrm.2012.08.014.

Acknowledgements

The authors thank Dr. Raj Bhatnagar, Dr. Manzar J. Hossain,Dr. Renu Tuteja and Dr. Arun Pradhan for their critical comments. Wealso thank Piyush Khandelia and RimpaGhosh for their help in the gener-ation of yeast mutant strains. Authors further acknowledge Dr. DeeptiGangwar, Dr. Asif Mohmmed and Prof. V. S. Chauhan for their inputs re-garding PF13_0082 gene architecture. P.K.S. and S.K. acknowledge Councilof Scientific and Industrial Research, India for fellowships. C.D.J acknowl-edges Department of Biotechnology Research Associateship. This workwas supported by Department of Biotechnology, Government of India(BT/PR7523/BRB/10/484/2006).

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