SummaryRMgm-4589
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene tagging |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 30703164 |
MR4 number | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. yoelii |
Parent strain/line | P. y. yoelii 17XNL |
Name parent line/clone | Not applicable |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | Hart KJ, Lindner SE |
Name Group/Department | Department of Biochemistry and Molecular Biology, Center for Malaria Research |
Name Institute | Pennsylvania State University, University Park, State College |
City | Pennsylvania |
Country | USA |
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Name of the mutant parasite | |
RMgm number | RMgm-4589 |
Principal name | CCR4-1::GFP |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Using transgenic PyCCR4-1::GFP parasites, we observed that PyCCR4-1 localized to cytoplasmic puncta in asexual blood stage parasites, and is similarly localized in both male and female gametocytes. Moreover, this expression profile extends to oocysts, oocyst sporozoites, and salivary gland sporozoites, where PyCCR4-1 was seen both in cytosolic puncta and located diffusely throughout the parasite. However, PyCCR4-1 was not detected above background in liver stage parasites. |
Gametocyte/Gamete | Using transgenic PyCCR4-1::GFP parasites, we observed that PyCCR4-1 localized to cytoplasmic puncta in asexual blood stage parasites, and is similarly localized in both male and female gametocytes. Moreover, this expression profile extends to oocysts, oocyst sporozoites, and salivary gland sporozoites, where PyCCR4-1 was seen both in cytosolic puncta and located diffusely throughout the parasite. However, PyCCR4-1 was not detected above background in liver stage parasites. |
Fertilization and ookinete | Not tested |
Oocyst | Using transgenic PyCCR4-1::GFP parasites, we observed that PyCCR4-1 localized to cytoplasmic puncta in asexual blood stage parasites, and is similarly localized in both male and female gametocytes. Moreover, this expression profile extends to oocysts, oocyst sporozoites, and salivary gland sporozoites, where PyCCR4-1 was seen both in cytosolic puncta and located diffusely throughout the parasite. However, PyCCR4-1 was not detected above background in liver stage parasites. |
Sporozoite | Using transgenic PyCCR4-1::GFP parasites, we observed that PyCCR4-1 localized to cytoplasmic puncta in asexual blood stage parasites, and is similarly localized in both male and female gametocytes. Moreover, this expression profile extends to oocysts, oocyst sporozoites, and salivary gland sporozoites, where PyCCR4-1 was seen both in cytosolic puncta and located diffusely throughout the parasite. However, PyCCR4-1 was not detected above background in liver stage parasites. |
Liver stage | Using transgenic PyCCR4-1::GFP parasites, we observed that PyCCR4-1 localized to cytoplasmic puncta in asexual blood stage parasites, and is similarly localized in both male and female gametocytes. Moreover, this expression profile extends to oocysts, oocyst sporozoites, and salivary gland sporozoites, where PyCCR4-1 was seen both in cytosolic puncta and located diffusely throughout the parasite. However, PyCCR4-1 was not detected above background in liver stage parasites. |
Additional remarks phenotype | Mutant/mutation 'Here, it was found that PyCCR4-1, a member of the CAF1/CCR4/NOT RNA metabolic complex, acts upon transcripts both directly and indirectly in gametocytes, and results in a reduction of male gametocytemia. In gametocytes lacking PyCCR4-1, as well as those expressing a catalytically dead variant, the initial coordinated wave of male gametocyte activation is lost, and these parasites are ~4-fold less able to productively infect mosquitoes. We find that the deletion of the C-terminal portion of CAF1 in both Plasmodium yoelii and Plasmodium falciparum phenocopies the deletion of pyccr4-1. We also find that the CAF1/CCR4/NOT complex is directly binding some of these transcripts and is likely acting both directly upon mRNAs and indirectly to modulate transcript abundance' Evidence is presented that: 'To experimentally determine the composition of this complex in Plasmodium yoelii, a transgenic PyCCR4-1::GFP parasite was created. The CAF1/CCR4/NOT complex was immunoprecipitated via the GFP tag from synchronized schizonts, when PyCCR4-1 is most abundant and is most prominently localized to cytoplasmic granules. As seen in other eukaryotes, PyCCR4-1 associates directly or indirectly through bridging interactions with most members of the canonical CAF1/CCR4/NOT complex in P. yoelii. Specifically, through mass spectrometric analyses we found that PyCCR4-1 associates with CAF1, NOT1, CAF40, NOT2 and a NOT family protein above our most stringent SAINT (Significance Analysis of INTeractome) threshold (0.1), and with NOT5 using a less stringent threshold (0.1 to 0.35). A small number of peptide spectral matches for NOT4 were also observed, but were not sufficiently enriched to be confidently included. This low abundance of NOT4 is consistent with its known transient association with the CAF1/CCR4/NOT complex in other eukaryotes. We also found that PyCCR4-1 interacts with proteins involved in the nuclear pore complex and RNA export (e.g. karyopherin-beta 3, exportin-1, UAP56), proteins involved in translation initiation (e.g. eIF2A, EF-1, EIF3D, PABP), and translational repression (e.g. CELF2/Bruno, DOZI, CITH, PABP). All of these interactions are consistent with appreciated CAF1/CCR4/NOT protein-protein or protein-RNA-protein interactions in other eukaryotes.' 'In eukaryotes, CCR4 and CAF1 function while in association with the other members of the CAF1/CCR4/NOT complex and is found in nuclear and cytosolic granular structures. Because PyCCR4-1 lacks an obvious LRR domain by which it can associate with the rest of the complex, we used immunofluorescence and live fluorescence assays to further validate these interactions. First, using transgenic PyCCR4-1::GFP parasites, we observed that PyCCR4-1 localized to cytoplasmic puncta in asexual blood stage parasites, and is similarly localized in both male and female gametocytes. Moreover, this expression profile extends to oocysts, oocyst sporozoites, and salivary gland sporozoites, where PyCCR4-1 was seen both in cytosolic puncta and located diffusely throughout the parasite. However, PyCCR4-1 was not detected above background in liver stage parasites. Thus, the near constitutive expression and localization of PyCCR4-1 in cytoplasmic foci in Plasmodium resembles that of its orthologues in model eukaryotes. Next, using either full length PyCAF1::GFP or PyCAF1ΔC::GFP transgenic parasites, we observed a similar expression and localization pattern to that of PyCCR4-1::GFP. Moreover, colocalization of PyCAF1ΔC::GFP and PyNOT1 signals were observed. Together, these data further indicate that the truncated PyCAF1ΔC::GFP variant can remain associated with the rest of its complex and yet phenocopies these PyCCR4-1-associated effects.' |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_1237700 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0519500 | ||||||||||||||||||||||||||
Gene product | CCR4 domain-containing protein 1, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | CCR4-1 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | GFP | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | unknown | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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