Characterization of a nuclear transport factor 2-like domain-containing protein in Plasmodium berghei

Background Plasmodium lacks an mRNA export receptor ortholog, such as yeast Mex67. Yeast Mex67 contains a nuclear transport factor 2 (NTF2)-like domain, suggesting that NTF2-like domain-containing proteins might be associated with mRNA export in Plasmodium. In this study, the relationship between mRNA export and an NTF2-like domain-containing protein, PBANKA_1019700, was investigated using the ANKA strain of rodent malaria parasite Plasmodium berghei. Methods The deletion mutant Δ1019700 was generated by introducing gene-targeting vectors into the P. berghei ANKA genome, and parasite growth and virulence were examined. To investigate whether PBANKA_1019700 is involved in mRNA export, live-cell fluorescence imaging and immunoprecipitation coupled to mass spectrometry (IP-MS) were performed using transgenic parasites expressing fusion proteins (1019700::mCherry). Results Deletion of PBANKA_1019700 affected the sexual phase but not the asexual phase of malaria parasites. Live-cell fluorescence imaging showed that PBANKA_1019700 localizes to the cytoplasm. Moreover, IP-MS analysis of 1019700::mCherry indicated that PBANKA_1019700 interacts with ubiquitin-related proteins but not nuclear proteins. Conclusions PBANKA_1019700 is a noncanonical NTF2-like superfamily protein. Supplementary Information The online version contains supplementary material available at 10.1186/s12936-024-04839-9.


Background
Export of mRNA through nuclear pores into the cytoplasm is an essential process in all eukaryotic cells.The steps of mRNA export have mostly been studied in opisthokonts, such as yeast [1,2].Mature mRNA is recognized by adaptor proteins such as nuclear poly(A) binding protein 2 (NAB2), yeast RNA annealing protein (YRA1), and three serine/arginine-rich (SR) proteins, including nucleolar protein 3 (NPL3), G-strand binding protein 2 (GBP2) and hypothetical RNA-binding protein (HRB1) [1,3].The mRNA export receptor, Mex67/Mtr2, recruited by these adaptor proteins interacts with nuclear pore complex proteins to export mRNA into the cytoplasm [2,4,5].
Plasmodium is a unicellular eukaryote with non-photosynthetic plastids that belongs to the phylum Apicomplexa [6].Plasmodium possess orthologs of yeast adaptor proteins, but no orthologs of Mex67 and Mtr2 [7][8][9].The mRNA export receptor in plants remains undiscovered [10].The mechanism of mRNA export in eukaryotes other than opisthokonts, such as yeasts and humans, is currently unclear.
In a previous study, mRNA export receptor-associated protein was not detected by immunoprecipitation coupled to mass spectrometry (IP-MS) in Plasmodium berghei [7], but its existence was not definitively ruled out.An in silico analysis may yet identify it in malaria parasites.Yeast Mex67 contains a nuclear transport factor 2 (NTF2)-like domain [11].The NTF2-like domain is necessary for heterodimerization with Mtr2 and for interaction with FG nucleoporins in the nuclear pores [11,12].Based on these findings, an NTF2-like domaincontaining protein might be associated with mRNA export in Plasmodium.
In the PlasmoDB database (www.plasm odb.org/), three proteins containing NTF2-like domains, namely, PBANKA_1019700 (conserved Plasmodium protein), PBANKA_0922900 (mitochondrial import inner membrane translocase subunit TIM44), and PBANKA_1030300 (nuclear transport factor 2), have been annotated.PBANKA_1030300 and PBANKA_0922900 are essential to parasite survival during the erythrocytic stage.PBANKA_1030300 binds RanGDP and is associated with protein import to the nucleus from the cytoplasm [13].PBANKA_0922900 contains a Tim44-like domain, indicating that it localizes to the mitochondria, not to the nucleus of Plasmodium.PBANKA_1019700 remains poorly understood, but in P. berghei it might be the mRNA export receptor-associated protein.In the present study, this possibility was investigated by examining the role and localization of PBANKA_1019700 by gene deletion, live-cell fluorescence imaging, and IP-MS.

Mouse studies and ethics
Five-to six-week-old female C57BL/6J (B6) mice were purchased from CLEA Japan Inc. (Tokyo, Japan).The experiments were approved (#221) by the Experimental Animal Ethics Committee of Kyorin University School of Medicine (Tokyo, Japan), and all experimental animals were kept at the animal facility in a specific-pathogenfree unit with sterile bedding, food, and water.
The infection studies included frequent observations to determine humane endpoints, at which mice were unable to ambulate sufficiently to obtain water or food.At the indicated time points, mice were euthanized by cervical dislocation under isoflurane anesthesia.All experiments were designed to minimize suffering.When symptoms or death was expected due to experimental infections, mice were visually checked by investigators at least twice daily (including weekends and holidays).Mice that exhibited signs of neurological distress, such as cerebral paralysis, were humanely sacrificed by cervical dislocation under isoflurane anesthesia and scored as deaths.No mice died before meeting the criteria for euthanasia.The investigators who conducted the experiments had completed the Experimental Animal Ethics Committee training course on animal care and handling.

Parasites and infection
Malaria parasites were stored as frozen stocks in liquid nitrogen.Luciferase-expressing P. berghei (strain ANKA) were generated as previously described [14,15].Erythrocytes parasitized with transfected parasites were generated in donor mice inoculated intraperitoneally with frozen stocks of parasites.The donor mice were monitored for parasitaemia daily and bled for experimental infection during periods in which the level of parasitaemia increased (1-2% parasitaemia).Experimental mice were infected intravenously with 1 × 10 4 parasitized erythrocytes or 5 × 10 6 to 5 × 10 7 purified mature schizonts harvested by Nycodenz density gradient centrifugation of infected blood from a given parasite strain.

Transfection
To generated deletion mutants of P. berghei ANKA, the gene-targeting vectors for PBANKA_1019700, PBANKA_1101300 (sbp1) and PBANKA_0519900 were designed and constructed as shown in Additional file 1: Fig. S1.Briefly, the 5′ and 3′ flanking regions of the open reading frame (ORF) of target genes were amplified by PCR.The PCR products were annealed to either side of the human dihydrofolate reductase (hdhfr)-expressing cassette, the red fluorescent protein gene (mCherry)hdhfr-expressing cassette or the luciferase gene (luc2)hdhfr-expressing cassette and amplified by PCR using gene-specific primers (Additional file 1: Fig. S1 and Additional file 2: Table S1) as previous studies [16,17].The gene-targeting vectors were introduced into the 5′ and 3′ flanking regions of target genes by double-crossover homologous recombination.To generate transgenic parasites expressing mCherry-fused PBANKA_1019700, the gene-targeting vectors for PBANKA_1019700 were prepared by PCR (Additional file 1: Fig. S1).The PCR products were annealed to either side of the red fluorescent protein gene (mCherry)-hdhfr-expressing cassette and amplified by PCR using gene-specific primers (Additional file 1: Fig. S1 and Additional file 2: Table S1).The gene-targeting vectors were introduced into the 3′ flanking regions of target genes by double-crossover homologous recombination.
To generate transgenic parasites expressing GFP-fused PBANKA_0519900 and GFP-fused PBANKA_1359300, the gene-targeting vectors for PBANKA_0519900 and PBANKA_1359300 were prepared by PCR, respectively (Additional file 1: Fig. S1).The PCR products were annealed to either side of the green fluorescent protein gene (gfp)-mutated human deoxyhypusine synthase (hdhps)-expressing cassette [18] and amplified by PCR using gene-specific primers (Additional file 1: Fig. S1 and Additional file 2: Table S1).The gene-targeting vectors were introduced into the 3′ flanking regions of ORFs of target genes by double-crossover homologous recombination.Transfection was performed using an Amaxa Basic Parasite Nucleofector Kit (Amaxa GmbH, Cologne, Germany) according to the manufacturer's protocol [19].

Genomic PCR
To generate gene-targeting vectors and confirm the introduction of gene-targeting vectors into target genes, genomic PCR was performed as described previously [7].Thirty-five cycles of PCR were performed on a C1000 thermal cycler (Bio-Rad, Hercules, CA, USA).Each cycle consisted of denaturation at 98 °C for 15 s, annealing at 55 °C for 15 s, and extension at 68 °C for 1-6 min.The PCR products were then analysed on a 1% (w/v) agarose gel and stained with ethidium bromide.

Parasitaemia
Methanol-fixed tail-blood smears, stained with 3% Giemsa diluted with phosphate buffer (pH 7.2) for 45 min, were subjected to microscopic examination.The number of parasitized erythrocytes (out of 250 erythrocytes) was enumerated when the level of parasitaemia and gametocytaemia exceeded 10%, while 1 × 10 4 erythrocytes were examined in mice with lower levels of parasitaemia and gametocytaemia.The parasitaemia and gametocytaemia percentage values were calculated as follows: [(number of parasitized erythrocytes) ÷ (total number of erythrocytes)] × 100.

Evaluation of gametocyte production in vitro
To evaluate gametocyte production, early trophozoite stage malaria parasites were obtained from B6 mice exhibiting 1-2% parasitaemia.Parasitized erythrocytes were incubated for 22 h, 28 h, 45 h in a 12-well plate.Methanol-fixed blood smears, stained with 3% Giemsa diluted in phosphate buffer (pH 7.2) for 45 min, were subjected to microscopic examination.Erythrocytes parasitized with mature schizonts containing 4-15 merozoites, and mature gametocytes showing sex-specific features such as nuclear enlargement, were counted as described previously [7,17].The distribution of pigment granules throughout the cytoplasm, and enlargement of cells, were also assessed [7,17].The proportions of male and female gametocytes were determined in at least 300 parasitized erythrocytes.The proportions of male and female gametocytes were calculated as follows: [(number of male or female gametocytes) ÷ (total number of schizonts plus male and female gametocytes)] × 100.

Ex vivo organ bioluminescence imaging
Bioluminescence imaging was performed using a Photon IMAGER system (Biospace Lab, Nesles-la-Vallée, France) as previously described [15].Mice were administered 1.5 mg of VivoGlo ™ Luciferin (In Vivo Grade, Promega, Japan) dissolved in 150 µL of phosphate buffered saline by intravenous injection.At 15 min after receiving the VivoGlo ™ Luciferin, organs were collected for image acquisition within 5 min.A charge-coupled device camera was used to monitor the acquisition of emitted photons.Ex vivo bioluminescence imaging data were analysed using the M3 software (Biospace) with size-constant regions of interest (ROIs).

Fluorescence live cell imaging
Nuclear DNA was stained using Hoechst 33342 dye (Invitrogen, Waltham, MA).To examine the localization of PBANKA_1019700::mCherry, Hoechst 33342 was added to the culture of parasitized erythrocytes at a concentration of 1 µg/mL.The staining medium was removed after the incubation, and fresh RPMI1640 medium was added.Brightfield and fluorescence micrographs were captured at 1000× magnification using an All-in-One Fluorescence Microscope (BZ-X800; KEYENCE Japan, Osaka, Japan).

Protein IP
Parasitized erythrocytes were transferred to RPMI1640 medium supplemented with 25% fetal bovine serum, 0.05 mg/mL penicillin and 0.05 mg/mL streptomycin.The parasitized erythrocytes were incubated for 22 h in 90% N 2 , 5% CO 2 and 5% O 2 .Mature schizonts and gametocytes were harvested by Nycodenz density gradient centrifugation, as described previously [7].Proteins were extracted using Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's protocol.Protein IP in transgenic parasites expressing mCherry fused to PBANKA_1019700 was performed using RFP-Trap Agarose and a RFP-Trap-A kit, according to the manufacturer's instructions (Chromotek, Planegg, Germany).

Mass spectrometry
All of the fractionated peptides obtained according to the manufacturer's instructions of RFP-Trap-A kit were injected into a trap column (C18, 75 μm × 2 cm; Acclaim PepMap 100, Thermo Fisher Scientific) and an analytical column (C18, 0.075 × 120 mm; Nikkyo Technos, Tokyo, Japan), which was attached to a nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) system.The nanoLC-MS/MS analysis was conducted using an Q Exactive plus mass spectrometer (Thermo Fisher Scientific) equipped with a nanoLC interface and a nano high-performance liquid chromatography (nanoHPLC) system (EASY-nLC 1200, Thermo Fisher Scientific).Purified peptides from the nanoLC were introduced into the Q Exactive plus, a hybrid quadrupole Fourier transform mass spectrometer.Full MS and MS/MS scans were followed by higher energy collisionally activated dissociation (HCD).The database search engines Proteome Discoverer 1.4 (Thermo Scientific) and MASCOT 2.6 (Matrix Science) were used to identify and quantify proteins from the MS, MS/MS and reporter ion spectra of the peptides.Peptide mass data were matched by searching the protein database (PlasmoDB-59_PbergheiANKA. fasta), downloaded from PlasmoDB (updated August 22, 2022).The false discovery rate (FDR) [20] was calculated by peptide sequence analysis using Percolator software [21].High-confidence peptide identifications were obtained by setting a target false discovery rate threshold of ≤ 1.0% at the peptide level.Proteins exhibiting at least three peptide spectral matches were included.

Statistical analysis
For time-series comparisons, Student's t test was performed using Statcel program (OMS, Saitama, Japan).Survival curves were compared using a log-rank test.P-values < 0.05 were considered statistically significant.

The NTF2 domain-containing protein PBANKA_1019700 is not associated with asexual development in malaria parasites
To investigate the relationship between mRNA export and PBANKA_1019700, deletion mutants (Δ1019700) were generated by introducing gene-targeting vectors into the P. berghei ANKA genome (Additional file 1: Fig. S1A).The course of parasitaemia in mice infected with Δ1019700 mutants was comparable to that in mice infected with control parasites (Fig. 1A).On day 8 post-infection, all mice infected with Δ1019700 mutants died, which was comparable to mice infected with control parasites (Fig. 1B).To examine the effects of PBANKA_1019700 deletion on the cell cycle during the asexual phase, mice were injected with schizonts of the control parasites and Δ1019700 mutants.At 22 h after schizont injection, ring stage-infected erythrocytes were observed in mice treated with both the control and Δ1019700 parasites (Fig. 1C).
Schizonts of malaria parasites bind to vascular endothelial cells through interactions between the endothelial receptor CD36 and sequestration-related proteins such as skeleton binding protein 1 (SBP1) and membrane associated histidine-rich protein 1a (MAH-RP1a) [22].To investigate whether the accumulation pattern of parasitized erythrocytes in the host is altered by PBANKA_1019700 deletion, bioluminescence imaging was performed (Fig. 1D, Additional file 1: Fig. S1B).The accumulation pattern of parasitized erythrocytes was not altered by PBANKA_1019700 deletion (Fig. 1D).Meanwhile, mutant P. berghei ANKA lacking SBP1 (Δsbp1), which was generated as a positive control, exhibited reduced sequestration in the lung and adipose tissues of mice (Fig. 1D, Additional file 1: Fig. S1C).Together, these results show that PBANKA_1019700 deletion does not affect the asexual development of malaria parasites.
Male and female gametocyte production is reduced in Δ1019700 mutants

Male and female gametocyte production is reduced in Δ1019700 mutants
To investigate the effects of PBANKA_1019700 deletion on gametocyte production in malaria parasites, erythrocytes parasitized with the control parasites and Δ1019700 mutants were cultured for 22, 28, or 45 h.The percentages of male and female gametocytes were lower in cultured Δ1019700 mutants than in control parasites (Fig. 2).These findings indicate that the NTF2 domaincontaining protein PBANKA_1019700 is involved in male and female gametocyte production in malaria parasites.

Identification of PBANKA_1019700-interacting proteins in malaria parasites
To investigate the proteins that interact with the NTF2 domain-containing protein PBANKA_1019700, protein immunoprecipitation (IP) using anti-mCherry beads was performed and the proteins bound to PBANKA_1019700 were identified through mass spectrometry (MS).IP-MS using anti-mCherry beads with wild-type P. berghei ANKA was performed as a control.
In three independent comparative proteomics analyses, 1244, 1304, and 1183 proteins were detected.Among them, 12 proteins with at least 50 peptide spectral matches (averaged from three independent experiments) and a fold change ≥ 5 compared to the control in three independent experiments were analysed further (Table 1 and Additional file 3: Table S2).Cytoplasmic proteins, including ubiquitin-related proteins (ubiquitin carboxylterminal hydrolase, PBANKA_0210600; ubiquitin-like protein, PBANKA_0823000; HECT-type E3 ubiquitin ligase UT, PBANKA_0802300) and a VPS13 domaincontaining protein (PBANKA_1359300) were detected through IP-MS of 1019700::mCherry (Table 1).However, RNA-binding proteins, such as NAB2 and GBP2, were not detected.Results are mean ± SD from three independent experiments.D Bioluminescence images of luciferase activity in the organs of infected mice.For bioluminescence analysis, erythrocytes parasitized with Pb ANKA were transferred to RPMI1640 medium supplemented with 25% fetal bovine serum, 0.05 mg/mL penicillin, and 0.05 mg/mL streptomycin.The parasitized erythrocytes were incubated for 18 h under 90% N 2 , 5% CO 2 , and 5% O 2 .Mature schizonts and gametocytes were harvested using Nycodenz density gradient centrifugation.B6 mice were injected with 5 × 10 6 -5 × 10 7 schizonts of luciferase-expressing P. berghei with p230 deletion (control), Δ1019700 or Δsbp1 mutants.At 22 h post-infection, d-luciferin (1.5 mg) was injected into the tail vein of each mouse and the organs from each group were removed after perfusion.Fold change indicates the change in luciferase activity compared to mice infected with luciferase-expressing P. berghei with p230 deletion (control).Dotted lines indicate significant differences (≥ twofold or ≤ 0.5-fold).Results are expressed as mean ± SD from three mice.Experiments were performed in duplicate with similar results

The conserved Plasmodium protein PBANKA_0519900 is associated with asexual development in malaria parasites
The conserved Plasmodium protein PBANKA_0519900 was detected through IP-MS of 1019700::mCherry.Although RMgmDB suggests that parasite growth is delayed by PBANKA_0519900 deletion, the roles and cellular localization of PBANKA_0519900 remain unclear.Therefore, deletion mutants (Δ0519900) were generated by introducing gene-targeting vectors into the P. berghei ANKA genome (Additional file 1: Fig. S1D) and the roles of PBANKA_0519900 in the asexual development of malaria parasites were examined.Mice infected with Δ0519900 mutants showed lower levels of parasitaemia than mice infected with control parasites (Fig. 4A) and their survival was prolonged compared to mice infected with control parasites (Fig. 4B).On the other hand, the percentages of both male and female gametocytes produced by Δ0519900 parasites were comparable to those of control parasites (Fig. 4C, D).Fig. 2 Effect of PBANKA_1019700 deletion on the sexual development of P. berghei ANKA.Erythrocytes parasitized with Plasmodium were incubated for 22 h, 28 h, and 45 h.The percentages of male and female gametocytes were calculated as follows: ([number of male or female gametocytes] ÷ [total number of schizonts plus male and female gametocytes]) × 100.A Percentages of mature female gametocytes.B Percentages of mature male gametocytes.Results are expressed as the mean ± SD from three independent experiments.*Significant difference compared to controls Table 1 Results of immunoprecipitation coupled with mass spectrometry for 1019700::mCherry parasites Proteins were extracted from 1019700::mCherry schizont-and gametocyte-enriched cultures after 22 h of culturing.Proteins with 50 peptide spectral matches (averaged across three independent experiments) and a fold change ≥ 5 compared to controls are listed.Control experiments involved immunoprecipitation of wildtype P. berghei ANKA using anti-mCherry beads coupled to mass spectrometry.Experiments were performed in triplicate.The results presented are sums from three independent experiments PSMs peptide spectral matches, AAs amino acids, MW molecular weight, Calc.pI calculated isoelectric point

Cellular localization of PBANKA_0519900 in malaria parasites
Next, to investigate the cellular localization of PBANKA_0519900, transgenic parasites expressing the fusion proteins 0519900::GFP (green fluorescent protein) and 1019700::mCherry were generated (Additional file 1: Fig. S3A).In addition, transgenic parasites expressing the fusion proteins 1359300::GFP and 1019700::mCherry were generated as cytoplasmic controls, as VPS13 domaincontaining proteins localize to the cytoplasm (Additional file 1: Fig. S3B).These GFP tags were introduced at the C-terminus of endogenous PBANKA_0519900 or PBANKA_1359300.Expression of 0519900::gfp and 1359300::gfp was controlled by the corresponding endogenous native promoters.Both transgenic parasites were successfully generated (Additional file 1: Fig. S3).
To examine the cellular localization of 0519900::GFP and 1359300::GFP, live-cell fluorescence imaging of trophozoites was performed.As shown in Fig. 5, GFP signals were present in the cytoplasm in 0519900::GFP/1019700::mCherryexpressing mutants.GFP signals were low levels in 1359300::GFP/1019700::mCherry-expressing mutants, but the signals were mainly present in the cytoplasm.These findings suggest that PBANKA_0519900 and PBANKA_1359300 are cytoplasmic proteins.Taken together, these results demonstrate that the NTF2 domain-containing protein PBANKA_1019700 is a noncanonical NTF2-like superfamily protein.

Discussion
The roles of PBANKA_1019700 during the asexual and sexual development stages of P. berghei ANKA were investigated using reverse genetics.The results showed that PBANKA_1019700 is not essential to either stage and is localized to the cytoplasm.Moreover, the IP-MS results for 1019700::mCherry indicated that PBANKA_1019700 does not interact with nuclear proteins.These results suggest that PBANKA_1019700 is not involved in mRNA export in malaria parasites.
In yeast, the Mex67/Mtr2 mRNA export receptor is required for mRNA export [1,2].Yeast Mex67 possesses an NTF2-like domain and interacts with adaptor proteins such as NAB2 [1,3].In Plasmodium, orthologs of Fig. 4 Effect of PBANKA_0519900 deletion on the asexual and sexual development of P. berghei ANKA.Female B6 mice were infected with 1 × 10 4 erythrocytes parasitized with P. berghei ANKA with PBANKA_0519900 deletion (Δ0519900).As a control, P. berghei ANKA with p230 deletion was inoculated intravenously into mice.*Significant difference compared to controls.A Time course of parasitaemia.Results are expressed as mean ± SD from four mice.Experiments using four mice were performed in triplicate.B Survival rate.Data are the mean ± SD from four mice and are representative of three independent experiments.C, D Percentages of mature female (C) and male (D) gametocytes.Erythrocytes parasitized with Plasmodium were incubated for 28 h.The percentages of male and female gametocytes were calculated as follows: ([number of male or female gametocytes] ÷ [total number of schizonts plus male and female gametocytes]) × 100.Results are expressed as the mean ± SD of three independent experiments adaptor proteins, such as NAB2, but not Mex67/Mtr2 have been identified.That PBANKA_1019700, which contains an NTF2-like domain, is the candidate protein of mRNA export receptor was supported by the results of the in silico analysis.
According to PlasmoDB, PBANKA_1019700 is expressed during the erythrocytic stage of the malaria parasite life cycle.However, the Rodent Malaria genetically modified Database (RMgmDB) contains no report on PBANKA_1019700 deletion.Meanwhile, the NTF2like domain and the C-terminus of PBANKA_1019700 are conserved among Plasmodium species, including Plasmodium falciparum.Data from transposon screening of P. falciparum indicate that PF3D7_1423700, which possesses an NTF2-like domain, is not essential to the asexual development of P. falciparum (PlasmoDB).
Similar to PF3D7_1423700, PBANKA_1019700 is not essential to the asexual development of P. berghei.Moreover, in this study, adaptor proteins such as NAB2 were not detected through IP-MS of 1019700::mCherry whereas live-cell imaging showed that PBANKA_1019700 is a cytoplasmic protein.In addition, previous research suggested that NAB2 derived from P. berghei (PbNAB2) does not interact with any NTF2-like superfamily proteins [7].
Trypanosomes belong to the Discoba, a clade outside of the Opisthokonta [6].In trypanosomes, an ortholog of yeast Mex67 has been characterized that is involved in mRNA export [23].However, no orthologs of adaptor proteins, such as yeast NAB2, have been identified through bioinformatics in trypanosomes [24].Although TbMex67 lacks the NTF2-like domain, it contains a CCCH-type zinc finger motif at its N-terminus [12].The CCCH-type zinc finger motif of TbMex67 is essential for its interaction with the nuclear pore [12].These findings indicate that an NTF2-like superfamily protein is not essential for mRNA export in parasitic unicellular eukaryotes.
In contrast to trypanosomes, orthologs of yeast Mex67 have not been identified in Plasmodium.However, Plasmodium possesses PbNAB2, which contains a CCCHtype zinc finger motif [7,8].In a previous study, IP-MS of PbNAB2::mCherry revealed that PbNAB2 interacts with PBANKA_1454600 [7], which was recently identified as the nucleoporin NUP269 [25], suggesting that PbNAB2 interacts directly with the nuclear pore to export mRNA.Together, these results indicate that proteins containing the CCCH-type zinc finger motif might directly interact with the nuclear pore and play a pivotal role in mRNA export in parasitic unicellular eukaryotes.NTF2-like superfamily proteins possess both enzymatic and non-enzymatic functions, including mRNA export.The noncanonical NTF2-like protein ActVI-ORFA has been suggested to play an enzymatic role in polyketide biosynthesis in Streptomyces [26].In this study, gametocyte production was reduced by PBANKA_1019700 deletion in P. berghei.IP-MS analysis of 1019700::mCherry showed that PBANKA_1019700 interacts with ubiquitin-related proteins and cytoplasmic PBANKA_0519900.In gametocytes, in which many biosynthetic pathways remain uncharacterized, the function of PBANKA_1019700 remains unclear.Our results thus far suggest that PBANKA_1019700 is a noncanonical NTF2-like superfamily protein associated with novel biosynthetic pathways, such as polyketide biosynthesis.

Conclusion
PBANKA_1019700 is a noncanonical NTF2-like superfamily protein and that export of mRNA is independent of NTF2-like superfamily proteins in Plasmodium.

Fig. 1
Fig.1Effect of PBANKA_1019700 deletion on the asexual and sexual development of Plasmodium berghei (strain ANKA).Female C57BL/6 (B6) mice were infected with 1 × 10 4 erythrocytes parasitized with Plasmodium berghei ANKA with PBANKA_1019700 deletion (Δ1019700).As a control, P. berghei ANKA with p230 deletion was inoculated intravenously into mice.A Time course of parasitaemia.Results are expressed as mean ± standard deviation (SD) from six mice.Experiments using six mice were performed in triplicate.B Survival rate.Data are the mean ± SD from six mice and are representative of three independent experiments.C Cell cycle during the asexual phase.Results are mean ± SD from three independent experiments.D Bioluminescence images of luciferase activity in the organs of infected mice.For bioluminescence analysis, erythrocytes parasitized with Pb ANKA were transferred to RPMI1640 medium supplemented with 25% fetal bovine serum, 0.05 mg/mL penicillin, and 0.05 mg/mL streptomycin.The parasitized erythrocytes were incubated for 18 h under 90% N 2 , 5% CO 2 , and 5% O 2 .Mature schizonts and gametocytes were harvested using Nycodenz density gradient centrifugation.B6 mice were injected with 5 × 10 6 -5 × 10 7 schizonts of luciferase-expressing P. berghei with p230 deletion (control), Δ1019700 or Δsbp1 mutants.At 22 h post-infection, d-luciferin (1.5 mg) was injected into the tail vein of each mouse and the organs from each group were removed after perfusion.Fold change indicates the change in luciferase activity compared to mice infected with luciferase-expressing P. berghei with p230 deletion (control).Dotted lines indicate significant differences (≥ twofold or ≤ 0.5-fold).Results are expressed as mean ± SD from three mice.Experiments were performed in duplicate with similar results