Plasmodium male gametocyte development and transmission are critically regulated by the two putative deadenylases of the CAF1/CCR4/NOT complex

Abstract

With relatively few known specific transcription factors to control the abundance of specific mRNAs, Plasmodium parasites may rely more on the regulation of transcript stability and turnover to provide sufficient gene regulation. Plasmodium transmission stages impose translational repression on specific transcripts in part to accomplish this. However, few proteins are known to participate in this process, and those that are characterized primarily affect female gametocytes. We have identified and characterized Plasmodium yoelii (Py) CCR4-1, a putative deadenylase, which plays a role in the development and activation of male gametocytes, regulates the abundance of specific mRNAs in gametocytes, and ultimately increases the efficiency of host-to-vector transmission. We find that when pyccr4-1 is deleted or its protein made catalytically inactive, there is a loss in the initial coordination of male gametocyte maturation and a reduction of parasite infectivity of the mosquito. Expression of only the N-terminal CAF1 domain of the essential CAF1 deadenylase leads to a similar phenotype. Comparative RNA-seq revealed that PyCCR4-1 affects transcripts important for transmission-related functions that are associated with male or female gametocytes, some of which directly associate with the immunoprecipitated complex. Finally, circular RT-PCR of one of the bound, dysregulated transcripts showed that deletion of the pyccr4-1 gene does not result in gross changes to its UTR or poly(A) tail length. We conclude that the two putative deadenylases of the CAF1/CCR4/NOT complex play critical and intertwined roles in gametocyte maturation and transmission.

Antagonistic roles of NOT1 paralogues in the timing of gene expression inPlasmodium falciparum

“…1E-H ). These cytosolic puncta did not overlap with aldolase or DAPI, whcih is consistent with previous reports for the P. yoelii orthologue, PyCCR4 [18] and the other three PfCCR4 paralogues [39]. This intracellular localization is consistent with other eukaryotic species suggesting the function of PfCCR4-NOT complex in posttranscriptional regulation via interactions with mRNA in the parasite cytoplasm.…”

“…The co-immunoprecipitation studies confirmed physical interaction of PfCAF40 with PfNOT1.1, PfNOT1.2, PfCAF1 and PfNOT4; PfCAF1 with PfCCR4; and either of the PfNOT1.1 or PfNOT1.2 with PfCAF40 and PfCAF1, respectively. On the other hand PfNOT4 failed to co-immunoprecipitate PfNOT1.1 and PfNOT1.2 (Table 1, top panel) , which is consistent with the transient character of this subunit with the CCR4-NOT complex previously demonstrated in Drosophila [52], human [53] and P.yoelii [18]. However, our data showed that PfNOT4 does associate with the PfCCR4-NOT complex given its interaction with CAF40 and possibly CAF1.…”

“…The existence of one copy of NOT1 as the scaffold of the CCR4-NOT complex has been reported in yeast [58], Drosophila [59], human cells [25] and Trypanosoma brucei , a parasitic kinetoplastid belonging to the Euglenozoa phylum [60]. Two copies of PfNOT1-encoding genes in P. falciparum is contrasting the majority of eukaryotes but is consistent with other Plasmodium species, such as P. yoelii [18], making it a unique feature in the Plasmodium genus. The presence of two copies of NOT1 is the likely result of gene duplication with the insertions of unique amino acid stretches in PfNOT1.2 MIF4G and NOT1 domains evolving with time.…”

“…This is supported by the abundant presence RNA-binding proteins [13, 14] and RNA degradation components [15] in the P. falciparum genome. The includePfALBA1 [16], PfCAF1 [17] in P. falciparum and PyCCR4 [18], PyALBA4 [19] in rodent-specific P.yoelii all of which functions were directly implicated in regulation of transcript levels. Moreover, variability of mRNA half-lives across the IDC observed by several experimental approaches also suggested a significant contribution of mRNA decay in regulation of gene expression in malaria parasites [20].…”

“…Initially it was shown that targeted gene disruption of PfCaf1 affected expression of 1,031 genes, including many invasion-related genes, which leads to the release of premature merozoites in P. falciparum [17]. In P. yoelii , PyCCR4 appears to regulate gene expression in gametocytes while disruption of PyCaf1 or PyCcr4 results in a reduction of male gametocyte activation [18]. Moreover, PyCCR4 was show to associate with some transcripts that are dysregulated in the parasite lacking PfCcr4 A potentially via its intrinsic DNA nuclease activity that is dependent on Cu 2+ [39].…”