PfDOZI在恶性疟原虫红内期生物学功能及转录后调控机制的研究

Malaria is major parasitic diseases that pose a severe threaten to human health. The researches on gene regulation mechanisms on plasmodium parasites are contribute to find the new drug targets. DOZI is an important RNA helicase in plasmodium parasites, which is highly conserved in plasmodium species with functional domains. However, pfdozi has been inadequately studied in intra-erythrocytic cycle, up to this point. The previous study indicated that pfdozi interrupt parasites showed higher blood stage replication rate when the parasitemia was detected. We hypothesized that PfDOZI may involved in mRNA metabolism and gene expression regulation and have an effect on the parasite growth in intra-erythrocytic stage. In order to characterize the biological function of PfDOZI, phenotype assays will be designed to address during intra-erythrocytic development. Additionally, RNA-seq will identify the effects of pfdozi disruption on transcriptome of parasites. To further explore the post-transcriptional regulation mechanism of PfDOZI, tandem affinity purification assay and RNA-IP will be used to uncover protein-protein interactions and its mRNA targets. Therefore, by discovering more about gene expression regulation of PfDOZI, we can provide basis for potential in vaccine and drug development.

疟疾是严重威胁人类健康的感染性寄生虫疾病，疟原虫基因表达调控机制研究有助于探索新型抗疟药物作用靶点。DOZI是疟原虫中重要的RNA解旋酶，高度保守且含有重要蛋白结构域。目前pfdozi基因在恶性疟原虫红内期阶段的功能尚未阐明。前期研究表明pfdozi基因敲除会导致红内期感染率升高。我们提出假设：PfDOZI作为RNA解旋酶，可能参与mRNA代谢过程，调控基因表达，影响疟原虫红内期阶段的生长发育。本项目拟从多方面观察pfdozi基因敲除引起的红内期阶段表型变化，通过RNA-seq技术分析pfdozi基因敲除对恶性疟原虫转录组的影响，探索PfDOZI的转录后调控机制。此外，本项目拟利用串联亲和纯化法和RNA-IP技术，明确PfDOZI的相互作用蛋白网络及其结合的靶向mRNA成分，进一步阐明其基因表达调控机制。通过以上一系列研究，以期为控制和消灭疟疾提供新的思路和理论依据。

疟疾是疟原虫经媒介按蚊传播的一种严重威胁人类健康的感染性寄生虫疾病。研究表明，基因的转录后调控机制对疟原虫的发育和分化至关重要，甚至会影响其阶段转化。本项目以恶性疟原虫DDX6-DEAD box解旋酶的同源物PfDOZI蛋白为出发点，解析其在疟原虫基因转录后调控过程中的生物学功能。研究发现pfdozi功能缺失可导致恶性疟原虫红内期无性阶段毒力增强，其原因可能是由于Δpfdozi虫株的红细胞入侵能力增强所致。同时，结果表明pfdozi基因敲除后配子体生成和发育过程中虫体形态均显著受到影响。其次，本课题运用RNA-seq技术对Δpfdozi虫株进行转录组分析，发现裂殖体阶段入侵相关基因的转录水平增强，但参与配子体发生的重要转录因子AP2-G却显著降低。并且，在Δpfdozi虫株配子体中众多有性阶段特异表达基因的mRNA水平显著下调，这些结果与表型观察结果一致。本项目进一步通过Pull-down实验联合LC-MS/MS技术揭示不同生长阶段PfDOZI的相互作用蛋白复合体成分，结果提示PfDOZI在裂殖体中主要形成P小体样mRNPs，发挥RNA降解作用；而在配子体中主要以应激颗粒样mRNPs为主，发挥翻译抑制作用。综上，我们认为PfDOZI蛋白在恶性疟原虫红内期不同生长阶段很可能通过与不同mRNPs成分发生相互作用，对其靶mRNA执行不同的转录后调控来维持疟原虫mRNA的稳态以完成阶段转化和环境适应。本研究为揭示疟原虫生物学特征和基因表达调控机制提供新的思路和实验依据。