Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of of CCR4-1

Protein (function)
In addition to transcript stabilization, translational control can also be accomplished by the degradation of transcripts. Degradation of mRNAs is typically initiated by deadenylases, which remove the protective poly(A) tail.
In many eukaryotes, the main complex responsible for deadenylation is the CAF1/CCR4/NOT complex, which also participates in transcriptional elongation, translational repression, and histone modification functions, and thus acts broadly upon gene expression.
To first assess the importance of the CAF1/CCR4/NOT complex in Plasmodium, we bioinformatically identified the genes for all members of the canonical CAF1/CCR4/NOT complex in Plasmodium, except for not3 and caf130. The absence of these two particular genes is not surprising, as these genes are also absent in some eukaryotes. In addition, we identified four CCR4 domain-containing proteins (PyCCR4-1, PyCCR4-2, PyCCR4-3, PyCCR4-4) that have homology to CCR4 deadenylases in other eukaryotes (e.g. yeast, human, mouse).
The typical domain architecture of CCR4-like proteins involves a Leucine Rich Repeat Region (LRR) and an Endonuclease/Exonuclease/Phosphatase (EEP) domain. The LRR mediates the interaction of CCR4 with CAF1 and the rest of the NOT complex, while the EEP domain contains active site residues required for deadenylation activity. Of these, we found that the overall length and sequence conservation within the EEP domain of PyCCR4-1 aligns most closely with the consensus CCR4 domain-containing proteins from model eukaryotes and humans. However, beyond the CCR4-EEP domain, there is no significant homology between other regions from PyCCR4-1, 2, 3, and 4 to each other, or to homologues from model species.

Phenotype
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 information
From the paper:
'We obtained clonal gene-deletion mutants for all four CCR4 genes which revealed that deletion of any one of these genes individually was not lethal in asexual blood stages. Moreover, deletion of pyccr4-2, pyccr4-3, or pyccr4-4 resulted in transgenic parasites that behaved as wild-type in all life cycle stages with respect to parasite numbers, prevalence of mosquito infection, and developmental timing/completion throughout the Plasmodium life cycle. Thus, CCR4-2, -3, and -4 may play redundant roles with one or more of the other CCR4-domain containing proteins'.

'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:
- PyCCR4-1 associates with a canonical CAF1/CCR4/NOT complex
- The putative catalytic residues of PyCCR4-1 are required for its roles in gametocytogenesis and transmission
- Truncation of PyCAF1 phenocopies the deletion of pyccr4-1
- PyCCR4-1, PyCAF1, and PyNOT1 localize to discrete cytosolic granules
- PyCCR4-1 affects important gametocyte and mosquito stage transcripts

'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.'

Other mutants