RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. yoelii
DisruptedGene model (rodent): PY17X_0716700; Gene model (P.falciparum): PF3D7_0414500; Gene product: male development protein MD5, putative (RBPm1; RNA-binding protein (RBP))
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 5 June 2024, 18:22
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 38824128
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherGuan J, Yuan J
Name Group/DepartmentState Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network, School o
Name InstituteXiamen University
Name of the mutant parasite
RMgm numberRMgm-5408
Principal name∆Rbpm1
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal production of male and female gametocytes. However, no production of male gametes (exflaggelation). Normal female gamete production.
Fertilization and ookineteNormal production of male and female gametocytes. However, no production of male gametes (exflaggelation). Normal female gamete production. No ookinete formation
OocystNo ookinete, oocyst or sporozoite formation.
SporozoiteNo oocyst or sporozoite formation.
Liver stageNot tested
Additional remarks phenotype

The mutant lacks expression of Rbpm1

Protein (function)
The P. yoelii Rbpm1 gene encodes a protein of 361 amino acid (aa) residues, with two RNA recognition motifs (RRM1 and RRM2)

Normal production of male and female gametocytes. However, no production of male gametes (exflaggelation). Normal female gamete production. No ookinete, oocyst or sporozoite formation.

Additional information
Analysis of a mutant expressing a C-terminal 6xHA-tagged version of RBPm1 (RMgm-5409) or a C-terminal GFP-tagged version of Rbpm1 (RMgm-5410) showed the following:
- Immunofluorescent assay (IFA) of RBPm1::6xHA showed that RBPm1 was expressed only in gametocytes, but not in asexual blood stages, ookinetes, oocysts, and sporozoites. Gametocyte-specific expression of RBPm1 was also observed in Rbpm1::gfp in which the RBPm1 was tagged with GFP.
- To dissect whether RBPm1 is male-specific, the Rbpm1::6HA gametocytes were co-stained with antibodies against α-Tubulin (a male gametocyte marker) and HA. RBPm1 was only detectable in the male gametocytes.
Additionally, RBPm1 was tagged with 6HA (RMgm-5411) in the reporter line DFsc7 (RMgm-4477) which expresses mCherry in females and GFP in males and observed the male specific expression of RBPm1.
- Localization of Rbpm1 in males was nuclear and consistent protein abundance and nuclear localization of RBPm1 during gametogenesis was observed.

Analysis of mutants with the RNA recognition motifs RRM1 (119-190 aa) and RRM2 (203-274 aa) deleted of endogenous RBPm1 in the 17XNL (∆rrm1, RMgm-5412 and ∆rrm2, RMgm-5413) showed the same phenotype as ∆Rbpm1 indicating an essential role of both two RNA recognition motifs in RBPm1 function.

Genetic crosses were performed between ∆Rbpm1 mutant and the male-deficient line Δmap2 or the female-deficient line Δnek4. As expected, the cross between Δmap2 and Δnek4 produced the ookinetes in vitro. The ookinete formation was restored in the ΔRbpm1 parasites that were crossed with Δnek4 but not Δmap2, further confirming the ∆Rbpm1 defects in male gamete formation. 

Aberrant axonemes were formed in activated male gametocytes ΔRbpm1. All the axonemes in ΔRbpm1 showed severe defects with loss of either central singlet microtubules (MTs) or peripheral doublet MTs. Normal DNA synthesis in activated ΔRbpm1male gametocytes.

Evidence is presented that:
- RBPm1 deficiency causes defective intron splicing of axonemal genes (intron retention, IR, genes).
The whole part of intron was retained in the transcripts for most IR genes, while only a N-terminal part of intron was retained for three IR genes.
- All IR genes are male-specific and 8 IR genes are axoneme-related. Twelve out of the 26 genes were selected for further analysis, including 6 annotated (Kinesin8b, PF16, dhc6, dhc7, dlc1, dlc2) and 6 unannotated (PY17X_1109100, PY17X_0521800, PY17X_1311800, PY17X_1323900, PY17X_1357300, PY17X_1335600). Each gene was endogenously tagged at the N- or C-terminus with a 6HA in the 17XNL, generating the HA-tagged lines (Kinesin8b RMgm-5414, PF16 RMgm-5415, dhc6 RMgm-5416, dhc7 RMgm-5417, dlc1 RMgm-5418, dcl2 RMgm-5419, PY17X_1109100 RMgm-5420, PY17X_0521800 RMgm-5421, PY17X_1311800 RMgm-5422, PY17X_1323900 RMgm-5423, PY17X_1357300 RMgm-5424, PY17X_1335600 RMgm-5425). All 12 proteins  were specifically expressed in male gametocytes during parasite life cycle,  in agreement with their transcript profiling. In inactivated gametocytes, these proteins were distributed at cytoplasm, while after activation 11 of 12 proteins displayed axoneme  localization in the flagellating male gametes. These results suggested that RBPm1 controls intron splicing for a group of the axonemal genes.
- To analyze the effect of IR on the axonemal proteins after RBPm1 loss, the Rbpm1 gene was deleted in each of the following tagged lines: kinesin8B::6HA, PF16::6HA, dhc6::6HA, dlc1::6HA, dlc2::6HA, and 1109100::6HA obtaining 6 RBPm1-null lines. In the absence of RBPm1, all 6 6HA-tagged proteins were not detected or under detectable level in male gametocytes compared to the parental counterparts in both IFA and immunoblot. These results demonstrated that RBPm1 deficiency causes protein expression loss of target axonemal genes.
- To confirm the essential roles of P. yoelii Kinesin8B and PF16 in axoneme assembly (as reported in P. berghei), kinesin8b and PF16 genes were disrupted in the 17XNL, obtaining mutant lines Δkinesin8b (RMgm-5426) and ΔPF16 (RMgm-5427). Depletion of kinesin8b or PF16 blocked or severely impaired male gamete formation respectively. Both mutants produced no midgut oocyst in the infected mosquitoes. Ultrastructure analysis of activated male gametocytes revealed that the Δkinesin8b mutant failed to develop “9+2” axoneme with loss of both central and peripheral MTs, while most of the axonemes lost central MTs (shown as “9+0” or “9+1”) in the ΔPF16. Therefore, depletion of Kinesin8B or PF16 phenocopies RBPm1 deficiency in axoneme assembly.
- Intron retention leads to loss of axonemal protein in RBPm1-null male gametocytes
- Intron deletion restores axonemal proteins and partially rectifies axoneme assembly defects in RBPm1-null gametocytes
- RBPm1 interacts with spliceosome E complex and introns of axonemal genes
The endogenous RBPm1 was tagged with a HA::TurboID motif in the 17XNL, generating the line Rbpm1::TurboID (RMgm-5428) to identify RBPm1-interacting proteins in the gametocytes.
- RBPm1 directs splicing of axonemal introns inserted in a reporter gene
- RBPm1 directs splicing of axonemal introns inserted in an endogenous gene
Other mutants

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0716700
Gene Model P. falciparum ortholog PF3D7_0414500
Gene productmale development protein MD5, putative
Gene product: Alternative nameRBPm1; RNA-binding protein (RBP)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedCRISPR/Cas9 construct: integration through double strand break repair
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe CRISPR/Cas9 plasmid pYCm was utilized for gene editing. To construct plasmids for gene tagging, the 5’- and 3’- flanking sequences (300 to 700 bp) at the designed insertion site of target genes were amplified as homologous templates. DNA fragments encoding 6HA, GFP, or 4Myc placed between them and in-frame with the target gene. To construct plasmids for N-terminal tagging, left homologous arms comprised 300 to 700 bp sequences upstream of the start codon, while right homologous arms comprised 300 to 700 bp sequences of the upstreaming sequences of the target gene. A DNA fragment encoding HA or 4Myc was then inserted between the left and right homologous arms in-frame with the target gene.
To construct the plasmids for gene knockout, left and right homologous arms consisted of 400 to 700 bp sequences upstream and downstream of the coding sequences of the target gene. To construct plasmids for RRM or intron deletion, the RRM or intron and 200 to 700 bp sequences upstream and downstream of the RRM or intron were PCR-amplified and inserted into specific restriction sites in pYCm, followed by RRM or intron deletion using PCR-based site-directed mutagenesis with mutation primers. To construct the plasmids for inserting the intron into the gep1 gene, the left and right homologous arms were composed of gep1 coding sequences ranging from 300 to 600 bp upstream and downstream of the insertion site, respectively. The left homologous arm, intron, and right homologous arm were connected using overlap PCR. Subsequently, the fused fragment was inserted into specific restriction sites in pYCm. For each of the modifications mentioned above, at least two small guide RNAs (sgRNAs) were designed. Forward and reverse single sgRNA oligonucleotides were mixed, denatured at 95°C for 2 minutes, annealed at room temperature for 5 minutes, and ligated into the pYCm. To construct plasmids for the bfp reporter assay, the intact bfp reporter (717 bp) driven by the 5'-UTR (1755 bp) of the hsp70 gene and the 3'-UTR (561 bp) of the dhfr gene were inserted into specific restriction sites between the left and right homologous arms of the p230p gene deletion plasmid. The kinesin8b intron 1 (239 bp), kinesin8b intron 2 (148 bp), PF16 intron 1 (276 bp), dlc1 intron 4 (193 bp), PY17X_1109100 intron 1 (353 bp), and PY17X_1109100 intron 2 (272 bp) were inserted into the bfp reporter by overlap PCR.
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6