Mutant/mutation
The mutant lacks expression of NOT1-G and expresses GFPmut2. The gfpmut2 expression cassette is introduced into the not1-g locus; no information is provided on the promoter driving GFPmut2.

Protein (function)
'We identified a protein annotated only as “NOT Family Protein” (PY17X_1027900) that was found to be associated with CCR4-1. Bioinformatic analyses using PlasmoDB and BLASTp identified that the protein encoded by PY17X_1027900 mostly closely matched eukaryotic NOT1 proteins but lacked a bioinformatically predictable TTP-binding domain that is present in the gene currently annotated as NOT1 (PY17X_0945600). We concluded that these 2 not1 genes were paralogues due to the high degree of sequence conservation in specific domains at both the DNA and protein levels (DNA: CAF1-binding domain 72% identity, NOT4-binding domain 66% identity; Protein: CAF1-binding domain 41% identical/63% positive, CAF40-binding domain 33%/57%, NOT4-binding domain 48%/62%, NOT1 domain 50%/73%). Moreover, these genes are highly conserved, syntenic, and only present across Plasmodium species and 2 other species of the Aconoidasida class (Genera: Theileria, Babesia) but not in other apicomplexans, model eukaryotes, or humans.'

Phenotype
No significant asexual blood-stage growth defects were observed for py17x_0945600-null parasites until late in the blood-stage infection (day 10). At this point, there was a small but statistically significant decrease in peak parasitemia with a concomitant earlier time to parasite clearance. Evidence is presented for a slight increase in gametocyte production; no male gamete production (exflagellation); female gametes infertile (i.e. were unable to produce developing zygotes). No ookinete or oocyst production.

Additional information

Plasmodium’s NOT1 paralogues localize to cytosolic puncta: 'To determine if the 2 paralogues of NOT1 are used at different points in the Plasmodium life cycle, we used conventional reverse genetics approaches to append a C-terminal GFP tag to each NOT1 paralogue in P. yoelii  and assessed their expression and localization. Both NOT1 paralogues were expressed and localized at the same times in the life cycle, and the expression pattern for PyNOT1 and PyNOT1-G is cytosolic and at least partially punctate. This is consistent with the localization of other members of the CAF1/CCR4/NOT complex in Plasmodium, model eukaryotes, and human cells. Of note, the NOT1 paralogues were expressed in both asexual and sexual blood stage parasites with a nonuniform, cytosolic distribution with some puncta visible. Both paralogous proteins were expressed in mosquito stage parasites, with localization shifting from a cytosolic diffuse pattern in oocysts, to a more nucleus-proximal pattern in oocyst sporozoites, and, finally, to a more apical pattern in salivary gland sporozoites. No expression of either protein was observed in mid- or late-liver stage parasites. Together, these expression and staining patterns match what is commonly seen for members of the CAF1/CCR4/NOT complex in other eukaryotes, as well as what we observed previously in P. yoelii with 2 other members of this complex: CAF1 and CCR4-1 [21]. Coupled with proteomic data showing their association with CCR4-1, this strongly indicates that these NOT1 paralogues are resident members of the CAF1/CCR4/NOT complex in P. yoelii.

Evidence is presented that:
- Extensive transcriptomic dysregulation in pynot1-g⁻ schizonts and gametocytes
- The tristetraprolin-binding domain of PyNOT1-G is dispensable for its essential roles in blood stage parasites but is important for transmission

'In agreement with the critical/essential role of NOT1 to eukaryotic RNA metabolism, our attempts to delete py17x_1027900 were largely unsuccessful. However, in 1 of 2 technical duplicates of 6 independent transfection attempts, we were able to obtain a 100% transgenic population with an extremely slow growth phenotype. It was only possible to produce this parasite line because the population was entirely transgenic, as the presence of even a small number of wild-type parasites with de novo resistance to pyrimethamine would likely have rapidly outgrown these transgenic parasites in the mouse. These data align with results from both PlasmoGEM (P. berghei) and piggyBac (P. falciparum) genetic screens that noted the importance of this gene to asexual blood-stage growth. Due to the severe asexual blood-stage defect, which closely aligned with observations of NOT1-related phenotypes in other eukaryotes, we propose that this “NOT Family Protein” is truly the NOT1 protein of Plasmodium parasites. Finally, it is notable that in contrast to other eukaryotes, PyNOT1 can be deleted and indicates that it is not strictly essential.'

Other mutants