Identification of Plasmodium yoelii CCR4/CAF1/CAF1d Binding partners and total proteomics of Py17XNL by Immunoprecipitation/nano LC-MS/MS

The transmission of the malaria parasite between mosquitoes and mammals requires translational repression to ensure that only the proper proteins are expressed at the right time, while still allowing the parasite to prepare the mRNAs it will need for the next developmental stage. With relatively few known specific transcription factors (ApiAP2 family) that may specifically initiate gene transcription, Plasmodium parasites also regulate the stability and turnover of transcripts to provide more comprehensive gene regulation. The CAF1/CCR4/NOT complex has been shown in model organisms to be important for not only mRNA degradation, but also translational control through its deadenylases CAF1 and CCR4. However, few proteins that impose translational repression in Plasmodium sexual stages are known, and those that are characterized primarily affect female gametocytes. Therefore, we have characterized two deadenylases and have uncovered their roles in transmission. We have identified and characterized CCR4-1, which we show plays a role in activating male gametocytes, stabilizing transcripts in gametocytes, and regulating host-to-vector transmission. We find that when ccr4-1 is genetically deleted, there is a loss in the coordination of male gametocyte activation and a reduction in the ability of the parasite to productively infect the mosquito, which is independent of its effect upon male activation. Comparative RNA-seq shows that the deletion of ccr4-1 affects many transcripts that are translationally repressed in females, are related to male gamete function, and/or are important for early mosquito stage development. In contrast, we found that genetic deletion of the major deadenylase Caf1 is lethal. However, we observed that expression of only the N-terminal Caf1 domain is permissive, yet prevents proper complex assembly and phenocopies the ccr4-1 deletion. We therefore conclude that the general and transmission-specialized deadenylases of the CAF1/CCR4/NOT complex play critical and intertwined roles in parasite growth and transmission.

疟原虫在蚊子与哺乳动物之间的传播依赖翻译抑制（translational repression）机制，以确保仅在正确时机表达适宜蛋白质，同时使疟原虫能够为下一发育阶段储备所需的信使RNA（mRNA）。由于目前已知的可特异性启动基因转录的转录因子数量极少，仅存在ApiAP2家族（ApiAP2 family），疟原虫还通过调控转录本的稳定性与降解周转，实现更为全面的基因表达调控。模式生物研究证实，CAF1/CCR4/NOT复合物（CAF1/CCR4/NOT complex）不仅参与mRNA降解，还可通过其脱腺苷酶（deadenylases）CAF1与CCR4介导翻译调控。然而，目前在疟原虫有性阶段中发挥翻译抑制作用的蛋白仍鲜有报道，且已被表征的此类蛋白主要作用于雌配子体（female gametocytes）。因此，本研究对两种脱腺苷酶展开了系统表征，并揭示了它们在疟原虫传播过程中的关键作用。我们鉴定并表征了CCR4-1，实验证明其可激活雄配子体（male gametocytes）、稳定配子体内的转录本，并调控宿主向媒介蚊子的传播过程。研究发现，当ccr4-1基因被敲除后，雄配子体激活的协调性会丧失，且疟原虫有效感染蚊子的能力下降，该表型独立于其对雄配子体激活的调控作用。对比RNA测序（RNA-seq）结果显示，ccr4-1基因的敲除会影响诸多转录本，这些转录本或在雌配子体内处于翻译抑制状态、或与雄配子功能相关、或对蚊子早期阶段发育至关重要。与之相反，主要脱腺苷酶Caf1的基因敲除会导致虫体致死。但我们观察到，仅表达Caf1的N端结构域（N-terminal Caf1 domain）即可维持虫体存活，却会阻碍复合物的正常组装，并在表型上模拟ccr4-1基因敲除的效果。综上，本研究认为，CAF1/CCR4/NOT复合物的通用型脱腺苷酶与传播特异性脱腺苷酶，在疟原虫的生长与传播过程中发挥着关键且相互交织的调控作用。