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The Plasmodium falciparum CCCH Zinc Finger Protein ZNF4 Plays an Important Role in Gametocyte Exflagellation through the Regulation of Male Enriched Transcripts

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Hanhsen, Borja, Farrukh, Afia, Pradel, Gabriele, Ngwa, Che Julius·2022·Cells
DOI 10.3390/cells11101666PMC PMC9139750

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Abstract

CCCH zinc finger proteins (ZFPs) function mainly as RNA-binding proteins (RBPs) and play a central role in the mRNA metabolism. Over twenty seven CCCH-ZFPs are encoded in the genome of the human malaria parasite Plasmodium falciparum, the causative agent of malaria tropica. However, little is known about their functions. In this study, we characterize one member of the PfCCCH-ZFP named ZNF4. We show that ZNF4 is highly expressed in mature gametocytes, where it predominantly localizes to the cytoplasm. Targeted gene disruption of ZNF4 showed no significant effect in asexual blood stage replication and gametocyte development while male gametocyte exflagellation was significantly impaired, leading to reduced malaria transmission in the mosquito. Comparative transcriptomics between wildtype (WT) and the ZNF4-deficient line (ZNF4-KO) demonstrated the deregulation of about 473 genes (274 upregulated and 199 downregulated) in mature gametocytes. Most of the downregulated genes show peak expression in mature gametocyte with male enriched genes associated to the axonemal dynein complex formation, and cell projection organization is highly affected, pointing to the phenotype in male gametocyte exflagellation. Upregulated genes are associated to ATP synthesis. Our combined data therefore indicate that ZNF4 is a CCCH zinc finger protein which plays an important role in male gametocyte exflagellation through the regulation of male gametocyte-enriched genes.

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Figure 1 Domain architecture and protein expression of ZNF4: (a) Schematic of the ZNF4 domain structure. The 204 kDa protein contains three CCCH zinc finger domains indicated in red. The black line indicates the region used for the generation of the recombinant peptide. (b) Predicted 3D structure of ZNF4. The 3D structure of the protein was predicted using AlphaFold (https://alphafold.ebi.ac.uk/ accessed on 12 January 2022) [40,41]. Colours indicate the confidence level of prediction which ranged from blue (very high confidence) to orange (very low confidence). (c) Transcript expression of ZNF4 in the blood stages of P. falciparum. Complementary DNA was synthesized from rings (R), trophozoites (TZ), schizonts (SZ), immature (imGC), mature (mGC), and gametocytes at 30 min post-activation (aGC) and subjected to diagnostic PCR using ZNF4-specific primers. The expression levels of Pfama1 and Pfccp2 were used to verify asexual blood stage and gametocyte-specific expression. Samples lacking reverse transcriptase (-RT) were used as controls to check for any contamination with gDNA. Pffbpa was used as a loading control. (d) Localization of ZNF4 in different blood stages of P. falciparum. Anti-ZNF4 was used to immunolabel fixed samples of trophozoites (TZ), schizonts (SZ), and gametocytes (GC stage II to V) as well as of activated gametocytes (aGC) at 30 min post-activation (green). Asexual blood stages (trophozoites and schizonts) were visualized by labelling with Evans blue and gametocytes were visualized by using rabbit anti-Pfs230 (red); nuclei were highlighted by Hoechst nuclear stain 33342 (blue). Bar, 5 µm.
Figure 1 Domain architecture and protein expression of ZNF4: (a) Schematic of the ZNF4 domain structure. The 204 kDa protein contains three CCCH zinc finger domains indicated in red. The black line indicates the region used for the generation of the recombinant peptide. (b) Predicted 3D structure of ZNF4. The 3D structure of the protein was predicted using AlphaFold (https://alphafold.ebi.ac.uk/ accessed on 12 January 2022) [40,41]. Colours indicate the confidence level of prediction which ranged from blue (very high confidence) to orange (very low confidence). (c) Transcript expression of ZNF4 in the blood stages of P. falciparum. Complementary DNA was synthesized from rings (R), trophozoites (TZ), schizonts (SZ), immature (imGC), mature (mGC), and gametocytes at 30 min post-activation (aGC) and subjected to diagnostic PCR using ZNF4-specific primers. The expression levels of Pfama1 and Pfccp2 were used to verify asexual blood stage and gametocyte-specific expression. Samples lacking reverse transcriptase (-RT) were used as controls to check for any contamination with gDNA. Pffbpa was used as a loading control. (d) Localization of ZNF4 in different blood stages of P. falciparum. Anti-ZNF4 was used to immunolabel fixed samples of trophozoites (TZ), schizonts (SZ), and gametocytes (GC stage II to V) as well as of activated gametocytes (aGC) at 30 min post-activation (green). Asexual blood stages (trophozoites and schizonts) were visualized by labelling with Evans blue and gametocytes were visualized by using rabbit anti-Pfs230 (red); nuclei were highlighted by Hoechst nuclear stain 33342 (blue). Bar, 5 µm.CC BY · PubMed Central
Figure 2 Targeted gene disruption of ZNF4 and its effect on asexual blood stage replication and gametocytogenesis: (a) Schematic depicting the generation of ZNF4-KO via single crossover recombination-based gene disruption using selective linked integration targeted gene disruption (SLI-TGD). The vector pSLI-TGD was modified to contain a 601 bp sequence block (white box with red stripes) from near the 5′ end of the ZNF4 coding region (red box). The coding region was maintained in frame with a GFP protein coding region (green box), a 2A “skip” peptide (black box), and the Neo-R gene (grey) that provides resistance to the antibiotic G418. Arrows indicate the position of primers 1–4, used to detect integration of the pSLI-TGD vector. Asterisks indicate a stop codon. GFP, green fluorescent protein; hDHFR; human dehydrofolate reductase for resistance to WR99210; NeoR, neomycin-resistance; 2A, skip peptide. (b) Confirmation of vector integration for the ZNF4-KO parasites by diagnostic PCR using gDNA obtained from ZNF4-KO and WT-NF54. 5′-integration was detected using primers 1 and 4 (1164 bp) and 3′-integration using primers 3 and 2 (955 bp). Primers 3 and 4 were used to detect the presence of episome (965 bp), and primers 1 and 2 were used for WT control (1194 bp). (c) Confirmation of truncated ZNF4 tagged with GFP. Parasite lysates obtained from different stages of the ZNF4-KO parasite line were subjected to Western blotting using polyclonal mouse anti-GFP (estimated size 53 kDa). Lysate from WT mature gametocyte was used as negative control. Immunoblotting with mouse anti-Pf39 antisera (39 kDa) served as a loading control. (d) Verification of GFP expression in the ZNF4-KO parasites by live imaging. Live images of trophozoites (TZ) and gametocyte stage II (GCII) of the ZNF4-KO line detected GFP (green) in the parasite. Dic, differential interference contrast. Nuclei were counterstained with Hoechst 33342 (blue). Bar, 5 µm. (e) Asexual blood stage replication of the ZNF4-KO. Synchronized ring stage cultures of WT and ZNF4-KO with an initial parasitaemia of 0.25% were maintained in cell culture medium, and the parasitaemia was followed over a time-period of 0 to 96 h via Giemsa stained smears. The data are a representation of one of two experiments performed in triplicate (mean ± SD). For the second experiment, see Figure S2a. (f) Disruption of ZNF4 shows no effect in gametocytaemia. Following two rounds of synchronization, a culture of 5% of the ring stage parasites of the WT and ZNF4-KO was induced for gametocytogenesis and, the next day, the parasites were washed and grown with medium supplemented with 50 mM GlcNac to kill asexual blood stages for 5 days, then with normal medium until day 10 post-induction. The gametocytaemia was monitored by Giemsa-stained blood smears from day 5 post-induction. The result is a representative of one of two experiments (see Figure S2b for second experiment). (g) Gametocyte maturation in the ZNF4-KO. The development of gametocyte was compared between the WT and the ZNF4-KO by counting the gametocyte stages of 50 gametocytes at each time point in triplicate (See Figure S2c for second experiment). (h) Gametocyte morphology in the ZNF4-KO line. Giemsa-stained pictures of gametocyte stages of the ZNF4-KO and WT.
Figure 2 Targeted gene disruption of ZNF4 and its effect on asexual blood stage replication and gametocytogenesis: (a) Schematic depicting the generation of ZNF4-KO via single crossover recombination-based gene disruption using selective linked integration targeted gene disruption (SLI-TGD). The vector pSLI-TGD was modified to contain a 601 bp sequence block (white box with red stripes) from near the 5′ end of the ZNF4 coding region (red box). The coding region was maintained in frame with a GFP protein coding region (green box), a 2A “skip” peptide (black box), and the Neo-R gene (grey) that provides resistance to the antibiotic G418. Arrows indicate the position of primers 1–4, used to detect integration of the pSLI-TGD vector. Asterisks indicate a stop codon. GFP, green fluorescent protein; hDHFR; human dehydrofolate reductase for resistance to WR99210; NeoR, neomycin-resistance; 2A, skip peptide. (b) Confirmation of vector integration for the ZNF4-KO parasites by diagnostic PCR using gDNA obtained from ZNF4-KO and WT-NF54. 5′-integration was detected using primers 1 and 4 (1164 bp) and 3′-integration using primers 3 and 2 (955 bp). Primers 3 and 4 were used to detect the presence of episome (965 bp), and primers 1 and 2 were used for WT control (1194 bp). (c) Confirmation of truncated ZNF4 tagged with GFP. Parasite lysates obtained from different stages of the ZNF4-KO parasite line were subjected to Western blotting using polyclonal mouse anti-GFP (estimated size 53 kDa). Lysate from WT mature gametocyte was used as negative control. Immunoblotting with mouse anti-Pf39 antisera (39 kDa) served as a loading control. (d) Verification of GFP expression in the ZNF4-KO parasites by live imaging. Live images of trophozoites (TZ) and gametocyte stage II (GCII) of the ZNF4-KO line detected GFP (green) in the parasite. Dic, differential interference contrast. Nuclei were counterstained with Hoechst 33342 (blue). Bar, 5 µm. (e) Asexual blood stage replication of the ZNF4-KO. Synchronized ring stage cultures of WT and ZNF4-KO with an initial parasitaemia of 0.25% were maintained in cell culture medium, and the parasitaemia was followed over a time-period of 0 to 96 h via Giemsa stained smears. The data are a representation of one of two experiments performed in triplicate (mean ± SD). For the second experiment, see Figure S2a. (f) Disruption of ZNF4 shows no effect in gametocytaemia. Following two rounds of synchronization, a culture of 5% of the ring stage parasites of the WT and ZNF4-KO was induced for gametocytogenesis and, the next day, the parasites were washed and grown with medium supplemented with 50 mM GlcNac to kill asexual blood stages for 5 days, then with normal medium until day 10 post-induction. The gametocytaemia was monitored by Giemsa-stained blood smears from day 5 post-induction. The result is a representative of one of two experiments (see Figure S2b for second experiment). (g) Gametocyte maturation in the ZNF4-KO. The development of gametocyte was compared between the WT and the ZNF4-KO by counting the gametocyte stages of 50 gametocytes at each time point in triplicate (See Figure S2c for second experiment). (h) Gametocyte morphology in the ZNF4-KO line. Giemsa-stained pictures of gametocyte stages of the ZNF4-KO and WT.CC BY · PubMed Central
Figure 3 ZNF4-KO impairs male gametocyte exflagellation and parasite transmission in the mosquito: (a) Disruption of ZNF4 impairs male gametocyte exflagellation. Mature WT and ZNF4-KO gametocytes were activated in vitro and the number of exflagellation centres was counted in 30 fields in triplicate using the light microscope. Three independent experiments were performed, indicated in numbers 1 to 3. **, p < 0.01, Student’s t test. (b) Mosquito infectivity of ZNF4-KO. Enriched mature gametocytes of WT or the ZNF4-KO were fed to An. stephensi mosquitoes via standard membrane feeding assays. The number of oocysts per midgut were counted at day 7 post-infection in four independent experiments; ***, p < 0.001, **, p < 0.01; ns, not significant, Student’s t test. (c) Percentage of infected mosquitoes in WT versus ZNF4-KO in each standard membrane-feeding experiment; * p < 0.05, Student’s t test.
Figure 3 ZNF4-KO impairs male gametocyte exflagellation and parasite transmission in the mosquito: (a) Disruption of ZNF4 impairs male gametocyte exflagellation. Mature WT and ZNF4-KO gametocytes were activated in vitro and the number of exflagellation centres was counted in 30 fields in triplicate using the light microscope. Three independent experiments were performed, indicated in numbers 1 to 3. **, p < 0.01, Student’s t test. (b) Mosquito infectivity of ZNF4-KO. Enriched mature gametocytes of WT or the ZNF4-KO were fed to An. stephensi mosquitoes via standard membrane feeding assays. The number of oocysts per midgut were counted at day 7 post-infection in four independent experiments; ***, p < 0.001, **, p < 0.01; ns, not significant, Student’s t test. (c) Percentage of infected mosquitoes in WT versus ZNF4-KO in each standard membrane-feeding experiment; * p < 0.05, Student’s t test.CC BY · PubMed Central
Figure 4 Deregulation of gene expression following disruption of ZNF4: (a) Comparative transcriptomics of deregulated genes in the ZNF4-KO parasite line in rings and mature gametocytes. Total RNA was extracted from ring stage parasites as well as the mature gametocytes of the ZNF4-KO and the WT and were subjected to comparative transcriptomics by RNA sequencing. Deregulated genes with relative gene expression greater than 2-fold were considered significant. (b) Validation of RNA-Seq data by qRT. Transcript analysis for 5 upregulated genes and 3 downregulated in mature gametocyte, as identified by RNA-Seq, were validated by real-time RT-PCR. Transcript expression levels were calculated by the 2−ΔCt method; the threshold cycle number (Ct) was normalized with the Ct of the gene encoding seryl tRNA-ligase (PF3D7_0717700) as reference. Genes were considered upregulated when the changes between ZNF4-KO and WT sample were greater than 2-fold. (c,d) Summary of Gene Ontology functional analysis of mature gametocyte differentially expressed genes following ZNF4 disruption. GO enrichment analysis of deregulated genes was determined using PlasmoDB (https://plasmodb.org/plasmo/app, accessed on 1 January 2022). The most significantly (p < 0.05) enriched GO terms in the biological process, the cellular component, and the molecular function are presented. For the complete list, see Table S2. All adjusted statistically significant values of the terms were the absolute log10 values. GO, gene ontology. (e) Pie chart showing peak expression stage of deregulated genes. The peak expression of the downregulated as well as the upregulated genes were determined using the 7 stage RNA-Seq data [44]. (f) Heat map of top 30 downregulated genes and their sex-specific expression. Heat map representing patterns of the top 30 downregulated genes and their sex-specific expression pattern [45]. Heat map was constructed using TB tools [42].
Figure 4 Deregulation of gene expression following disruption of ZNF4: (a) Comparative transcriptomics of deregulated genes in the ZNF4-KO parasite line in rings and mature gametocytes. Total RNA was extracted from ring stage parasites as well as the mature gametocytes of the ZNF4-KO and the WT and were subjected to comparative transcriptomics by RNA sequencing. Deregulated genes with relative gene expression greater than 2-fold were considered significant. (b) Validation of RNA-Seq data by qRT. Transcript analysis for 5 upregulated genes and 3 downregulated in mature gametocyte, as identified by RNA-Seq, were validated by real-time RT-PCR. Transcript expression levels were calculated by the 2−ΔCt method; the threshold cycle number (Ct) was normalized with the Ct of the gene encoding seryl tRNA-ligase (PF3D7_0717700) as reference. Genes were considered upregulated when the changes between ZNF4-KO and WT sample were greater than 2-fold. (c,d) Summary of Gene Ontology functional analysis of mature gametocyte differentially expressed genes following ZNF4 disruption. GO enrichment analysis of deregulated genes was determined using PlasmoDB (https://plasmodb.org/plasmo/app, accessed on 1 January 2022). The most significantly (p < 0.05) enriched GO terms in the biological process, the cellular component, and the molecular function are presented. For the complete list, see Table S2. All adjusted statistically significant values of the terms were the absolute log10 values. GO, gene ontology. (e) Pie chart showing peak expression stage of deregulated genes. The peak expression of the downregulated as well as the upregulated genes were determined using the 7 stage RNA-Seq data [44]. (f) Heat map of top 30 downregulated genes and their sex-specific expression. Heat map representing patterns of the top 30 downregulated genes and their sex-specific expression pattern [45]. Heat map was constructed using TB tools [42].CC BY · PubMed Central

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Research works printed but not linked: 58

  1. Kuehn, 2010, The coming-out of malaria gametocytes, J. Biomed. Biotechnol., 2010, 976827, 10.1155/2010/976827 · DOI
  2. Bennink, 2016, The development of malaria parasites in the mosquito midgut, Cell. Microbiol., 18, 905, 10.1111/cmi.12604 · DOI
  3. Josling, 2015, Sexual development in Plasmodium parasites: Knowing when it’s time to commit, Nat. Rev. Microbiol., 13, 573, 10.1038/nrmicro3519 · DOI
  4. Kafsack, 2014, A transcriptional switch underlies commitment to sexual development in malaria parasites, Nature, 507, 248, 10.1038/nature12920 · DOI
  5. Sinha, 2014, A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium, Nature, 507, 253, 10.1038/nature12970 · DOI
  6. Shang, 2021, A cascade of transcriptional repression determines sexual commitment and development in Plasmodium falciparum, Nucleic Acids Res., 49, 9264, 10.1093/nar/gkab683 · DOI
  7. Yuda, 2020, Female-specific gene regulation in malaria parasites by an AP2-family transcription factor, Mol. Microbiol., 113, 40, 10.1111/mmi.14334 · DOI
  8. Li, 2021, Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes, EMBO Rep., 22, e51660, 10.15252/embr.202051660 · DOI
  9. Kaneko, I., Iwanaga, S., Kato, T., Kobayashi, I., and Yuda, M. (2015). Genome-Wide Identification of the Target Genes of AP2-O, a Plasmodium AP2-Family Transcription Factor. PLoS Pathog., 11. · DOI
  10. Modrzynska, 2017, A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle, Cell Host Microbe, 21, 11, 10.1016/j.chom.2016.12.003 · DOI
  11. Bennink, 2019, The molecular machinery of translational control in malaria parasites, Mol. Microbiol., 112, 1658, 10.1111/mmi.14388 · DOI
  12. Bennink, S., von Bohl, A., Ngwa, C.J., Henschel, L., Kuehn, A., Pilch, N., Weißbach, T., Rosinski, A.N., Scheuermayer, M., and Repnik, U. (2018). A seven-helix protein constitutes stress granules crucial for regulating translation during human-to-mosquito transmission of Plasmodium falciparum. PLoS Pathog., 14. · DOI
  13. Mair, G.R., Lasonder, E., Garver, L.S., Franke-Fayard, B.M.D., Carret, C.K., Wiegant, J.C.A.G., Dirks, R.W., Dimopoulos, G., Janse, C.J., and Waters, A.P. (2010). Universal Features of Post-Transcriptional Gene Regulation Are Critical for Plasmodium Zygote Development. PLoS Pathog., 6. · DOI
  14. Mair, 2006, Regulation of sexual development of Plasmodium by translational repression, Science, 313, 667, 10.1126/science.1125129 · DOI
  15. Miao, 2010, The Puf-family RNA-binding protein PfPuf2 regulates sexual development and sex differentiation in the malaria parasite Plasmodium falciparum, J. Cell Sci., 123, 1039, 10.1242/jcs.059824 · DOI
  16. Shrestha, 2016, The RNA-binding protein Puf1 functions in the maintenance of gametocytes in Plasmodium falciparum, J. Cell Sci., 129, 3144
  17. Ngwa, 2017, Transcriptional Profiling Defines Histone Acetylation as a Regulator of Gene Expression during Human-to-Mosquito Transmission of the Malaria Parasite Plasmodium falciparum, Front. Cell. Infect. Microbiol., 7, 320, 10.3389/fcimb.2017.00320 · DOI
  18. Ngwa, C.J., Kiesow, M.J., Orchard, L.M., Farrukh, A., Llinás, M., and Pradel, G. (2019). The g9a histone methyltransferase inhibitor BIX-01294 modulates gene expression during plasmodium falciparum gametocyte development and transmission. Int. J. Mol. Sci., 20. · DOI
  19. Ngwa, 2021, Zinc finger proteins of Plasmodium falciparum, Cell. Microbiol., 23, e13387, 10.1111/cmi.13387 · DOI
  20. Hajikhezri, Z., Darweesh, M., Akusjärvi, G., and Punga, T. (2020). Role of CCCH-Type Zinc Finger Proteins in Human Adenovirus Infections. Viruses, 12. · DOI
  21. Taylor, 1996, Mitogens stimulate the rapid nuclear to cytosolic translocation of tristetraprolin, a potential zinc-finger transcription factor, Mol. Endocrinol., 10, 140
  22. Pomeranz, 2010, The Arabidopsis tandem zinc finger protein AtTZF1 traffics between the nucleus and cytoplasmic foci and binds both DNA and RNA, Plant Physiol., 152, 151, 10.1104/pp.109.145656 · DOI
  23. Pomeranz, 2011, Putative molecular mechanisms underlying tandem CCCH zinc finger protein mediated plant growth, stress, and gene expression responses, Plant Signal. Behav., 6, 647, 10.4161/psb.6.5.15105 · DOI
  24. Ngwa, C.J., Scheuermayer, M., Mair, G.R., Kern, S., Brügl, T., Wirth, C.C., Aminake, M.N., Wiesner, J., Fischer, R., and Vilcinskas, A. (2013). Changes in the transcriptome of the malaria parasite Plasmodium falciparum during the initial phase of transmission from the human to the mosquito. BMC Genom., 14. · DOI
  25. Ifediba, 1981, Complete in vitro maturation of Plasmodium falciparum gametocytes, Nature, 294, 364, 10.1038/294364a0 · DOI
  26. Lambros, 1979, Synchronization of Plasmodium falciparum erythrocytic stages in culture, J. Parasitol., 65, 418, 10.2307/3280287 · DOI
  27. Birnbaum, 2017, A genetic system to study Plasmodium falciparum protein function, Nat. Methods, 14, 450, 10.1038/nmeth.4223 · DOI
  28. Wagle, P., Nikolić, M., and Frommolt, P. (2015). QuickNGS elevates Next-Generation Sequencing data analysis to a new level of automation. BMC Genom., 16. · DOI
  29. Dobin, 2013, STAR: Ultrafast universal RNA-seq aligner, Bioinformatics, 29, 15, 10.1093/bioinformatics/bts635 · DOI · Paper page
  30. Pertea, 2015, StringTie enables improved reconstruction of a transcriptome from RNA-seq reads, Nat. Biotechnol., 33, 290, 10.1038/nbt.3122 · DOI
  31. Liao, 2014, featureCounts: An efficient general purpose program for assigning sequence reads to genomic features, Bioinformatics, 30, 923, 10.1093/bioinformatics/btt656 · DOI · Paper page
  32. Love, 2014, Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2, Genome Biol., 15, 1, 10.1186/s13059-014-0550-8 · DOI · Paper page
  33. Livak, 2001, Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method, Methods, 25, 402, 10.1006/meth.2001.1262 · DOI · Paper page
  34. Salanti, 2003, Selective upregulation of a single distinctly structured var gene in chondroitin sulphate A-adhering Plasmodium falciparum involved in pregnancy-associated malaria, Mol. Microbiol., 49, 179, 10.1046/j.1365-2958.2003.03570.x · DOI · Paper page
  35. Wang, 2010, Identification of a major rif transcript common to gametocytes and sporozoites of Plasmodium falciparum, Malar. J., 9, 147, 10.1186/1475-2875-9-147 · DOI
  36. Kariuki, 1998, Plasmodium falciparum: Purification of the various gametocyte developmental stages from in vitro-cultivated parasites, Am. J. Trop. Med. Hyg., 59, 505, 10.4269/ajtmh.1998.59.505 · DOI
  37. Fivelman, 2007, Improved synchronous production of Plasmodium falciparum gametocytes in vitro, Mol. Biochem. Parasitol., 154, 119, 10.1016/j.molbiopara.2007.04.008 · DOI
  38. Varadi, 2022, AlphaFold Protein Structure Database: Massively expanding the structural coverage of protein-sequence space with high-accuracy models, Nucleic Acids Res., 50, D439, 10.1093/nar/gkab1061 · DOI · Paper page
  39. Chen, 2020, TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data, Mol. Plant, 13, 1194, 10.1016/j.molp.2020.06.009 · DOI
  40. Grant, C.E., and Bailey, T.L. (2021). XSTREME: Comprehensive motif analysis of biological sequence datasets. bioRxiv. · DOI
  41. López-Barragán, M.J., Lemieux, J., Quiñones, M., Williamson, K.C., Molina-Cruz, A., Cui, K., Barillas-Mury, C., Zhao, K., and Su, X. (2011). Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum. BMC Genom., 12. · DOI
  42. Lasonder, 2016, Integrated transcriptomic and proteomic analyses of P. falciparum gametocytes: Molecular insight into sex-specific processes and translational repression, Nucleic Acids Res., 44, 6087, 10.1093/nar/gkw536 · DOI
  43. Zhang, 2018, Uncovering the essential genome of the human malaria parasitePlasmodium falciparum by saturationmutagenesis, Science, 360, eaap7847, 10.1126/science.aap7847 · DOI
  44. Silvie, 2021, Pleiotropic Roles for the Plasmodium berghei RNA Binding Protein UIS12 in Transmission and Oocyst Maturation, Front. Cell. Infect. Microbiol., 11, 624945, 10.3389/fcimb.2021.624945 · DOI
  45. Aprea, I., Raidt, J., Höben, I.M., Loges, N.T., Nöthe-Menchen, T., Pennekamp, P., Olbrich, H., Kaiser, T., Biebach, L., and Tüttelmann, F. (2021). Defects in the Cytoplasmic Assembly of Axonemal Dynein Arms Cause Morphological Abnormalities and Dysmotility in Sperm Cells Leading to male Infertility. PLoS Genet., 17. · DOI
  46. Lindemann, 2010, Flagellar and ciliary beating: The proven and the possible, J. Cell Sci., 123, 519, 10.1242/jcs.051326 · DOI
  47. Stone, 2017, A Molecular Assay to Quantify Male and Female Plasmodium falciparum Gametocytes: Results From 2 Randomized Controlled Trials Using Primaquine for Gametocyte Clearance, J. Infect. Dis., 216, 457, 10.1093/infdis/jix237 · DOI
  48. Bradley, 2018, Predicting the likelihood and intensity of mosquito infection from sex specific plasmodium falciparum gametocyte density, Elife, 7, e34463, 10.7554/eLife.34463 · DOI
  49. Leslie, 2020, MNS1 variant associated with situs inversus and male infertility, Eur. J. Hum. Genet., 28, 50, 10.1038/s41431-019-0489-z · DOI
  50. Ta-Shma, A., Hjeij, R., Perles, Z., Dougherty, G.W., Abu Zahira, I., Letteboer, S.J.F., Antony, D., Darwish, A., Mans, D.A., and Spittler, S. (2018). Homozygous loss-of-function mutations in MNS1 cause laterality defects and likely male infertility. PLoS Genet., 14. · DOI
  51. Parkhurst, 1994, Sex determination and dosage compensation: Lessons from flies and worms, Science, 264, 924, 10.1126/science.8178152 · DOI
  52. Wang, 1994, The sex-lethal amino terminus mediates cooperative interactions in RNA binding and is essential for splicing regulation, Genes Dev., 8, 2072, 10.1101/gad.8.17.2072 · DOI
  53. Schmid, 2015, The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production, Cell Rep., 12, 128, 10.1016/j.celrep.2015.06.008 · DOI
  54. Green, 2002, Nab2p is required for poly(A) RNA export in Saccharomyces cerevisiae and is regulated by arginine methylation via Hmt1p, J. Biol. Chem., 277, 7752, 10.1074/jbc.M110053200 · DOI
  55. Kini, 2014, Cytoplasmic poly(A) binding protein C4 serves a critical role in erythroid differentiation, Mol. Cell. Biol., 34, 1300, 10.1128/MCB.01683-13 · DOI
  56. Miao, J., Fan, Q., Parker, D., Li, X., Li, J., and Cui, L. (2013). Puf mediates translation repression of transmission-blocking vaccine candidates in malaria parasites. PLoS Pathog., 9. · DOI
  57. Hirai, M., Maeta, A., Mori, T., and Mita, T. (2021). Pb103 Regulates Zygote/Ookinete Development in Plasmodium berghei via Double Zinc Finger Domains. Pathogens, 10. · DOI
  58. Fu, 2017, RNA-binding proteins in immune regulation: A focus on CCCH zinc finger proteins, Nat. Rev. Immunol., 17, 130, 10.1038/nri.2016.129 · DOI

Metadata

Authors
Hanhsen, B., Farrukh, A., Pradel, G., & Ngwa, C. J.
Affiliations
RWTH Aachen University (DE)
Corresponding author
Ngwa, Che Julius
ORCID
Venue
Cells
Publisher
MDPI AG
Published
2022-05-17
Volume
11
Issue
10
Pages
1666
ISSN
2073-4409 (ISSN-L)
DOI
10.3390/cells11101666
PMC
PMC9139750
OpenAlex
W4280578208
Cited by
27
References
3
Citations by year
2026: 4 · 2025: 4 · 2024: 5 · 2023: 11 · 2022: 2
FWCI
4.86
Type
journal article
Language
en
Open access
Licence
Locations
  • Cells (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • RWTH Publications (RWTH Aachen) (submittedVersion, other-oa)
Funders
  • the Deutsche Forschungsgemeinschaft (NG170/1-1, PR905/20-1, No 731060)
  • the German Academic Exchange Service (DAAD) (NG170/1-1, PR905/20-1, No 731060)
  • the European Union’s Horizon 2020 research and innovation program (NG170/1-1, PR905/20-1, No 731060)
Topics
  • Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Malaria Research and Control
  • Life Sciences › Immunology and Microbiology › Parasitology › Bird parasitology and diseases
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
Plasmodium falciparum, CCCH zinc finger proteins, male gametocytes, ATP production, mosquito-borne transmission, gene knockout, transcriptomics, RNA-binding proteins
MeSH terms
Animals, Cytoplasm, Malaria, Male, Culicidae, Plasmodium falciparum, Zinc Fingers

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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How to cite

Cite CoChat for the checks on this page, and the paper for its own findings.

This page
CoChat. (2026). Citation check: The Plasmodium falciparum CCCH Zinc Finger Protein ZNF4 Plays an Important Role in Gametocyte Exflagellation through the Regulation of Male Enriched Transcripts. https://app.cochat.ai/papers/10.3390/cells11101666
The paper
Hanhsen, B., Farrukh, A., Pradel, G., & Ngwa, C. J. (2022). The Plasmodium falciparum CCCH Zinc Finger Protein ZNF4 Plays an Important Role in Gametocyte Exflagellation through the Regulation of Male Enriched Transcripts. Cells. https://doi.org/10.3390/cells11101666