MEMBERS OF THE PLASMODIUM YOELII CAF1/CCR4/NOT COMPLEX ARE CRUCIAL OR ESSENTIAL FOR PROPER GAMETOCYTE DEVELOPMENT
Open Access
- Author:
- Hart, Kevin Joseph
- Graduate Program:
- Immunology and Infectious Diseases
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 30, 2018
- Committee Members:
- Scott Eugene Lindner, Dissertation Advisor/Co-Advisor
Scott Eugene Lindner, Committee Chair/Co-Chair
Paul Lee Babitzke, Committee Member
Joseph C. Reese, Committee Member
Liwang Cui, Outside Member
Manuel Llinas, Committee Member - Keywords:
- Plasmodium
Malaria
CCR4
NOT1
CAF1
Parasitology - Abstract:
- 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 generate the mRNAs it will need for the next developmental stage. With relatively few known sequence specific transcription factors that may regulate initiation of transcription, Plasmodium also regulates the stability and turnover of transcripts to provide more comprehensive gene regulation. In model eukaryotes, the CAF1/CCR4/NOT complex has been shown to be crucial for not only mRNA degradation, but also translational control through its deadenylases CAF1 and CCR4. These deadenylases are thought to act primarily through association with NOT1 of the CAF1/CCR4/NOT complex. I have identified and characterized CCR4-1 in Plasmodium yoelii, and found that it plays a role in the activation and development of male gametocytes, and regulates host-to-vector transmission. With the deletion of ccr4-1, 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 that is independent of its effect upon male activation. While gene deletion of the major deadenylase CAF1 is lethal, expression of the N-terminal CAF1 domain maintains survival, but prevents proper complex assembly and phenocopies the ccr4-1 deletion. Additionally, in stark contrast to virtually all other eukaryotes, I have bioinformatically identified that the Aconoidasida class of Apicomplexans encode two putative NOT1 proteins. Generally, NOT1 acts as the scaffold for the CAF1/CCR4/NOT complex. I found that neither NOT1 nor its paralog NOT1-G are essential for Plasmodium yoelii asexual blood stage development, but that NOT1-G is essential for complete gametocyte development. Moreover, pynot1-g- parasites only produce female/immature gametocytes that, by genetic cross experiments, are sterile and cannot be transmitted to mosquitoes. Comparative transcriptomics of parasites with a pynot1-g deletion show massive dysregulation of the female gametocyte transcriptome and implicate NOT1-G as an early regulator of RNA metabolism for gametocytogenesis. Thus, I demonstrated that CCR4-1, CAF1, and NOT1-G play intertwined roles in, and are essential for, proper gametocyte development.