Post-transcriptional regulation by CCCH-type zinc finger family of Plasmodium parasites

Post-transcriptional regulatory mechanisms, such as RNA translation, stabilization and degradation following transcriptional activation of expressed genes, are essential for orderly protein expression required for malaria parasite stage formation. This study, focuses on the TZF family, which has family molecules throughout the parasite life cycle and is highly conserved among Plasmodium species, in order to clarify the basic principle of the post-transcriptional regulatory mechanism common to each stage formation. By clarifying the functions of the TZF family using novel target RNA identification methods and innovative genome editing methods, the aim is to elucidate the common principle of the post-transcriptional regulatory mechanism by the TZF family in the formation of each stage. This year, the TRIBE method was established using the RNA editing enzyme ADAR, which is a novel target RNA identification method. The TRIBE method was established using DOZI, a female gametocyte RNA-binding protein, as a model. The study compared RIP-seq and TRIBE method, and showed that the simple TRIBE method has sufficient detection sensitivity. On the other hand, fluorescent protein-fused gene expression strains were created for two asexually growing TZFs. In the preliminary analysis before the start of the research, a GFP-fused gene expression strain was created and fluorescence observation was performed. A fusion gene expression strain of mNeonGreen (mNG) with brighter fluorescence was generated for these two TZFs. Fluorescence observation revealed that both TZFs were expressed in all erythrocyte stages and localized in the perinuclear and cytoplasmic regions, respectively. In the case of the GFP fusion gene, fluorescence was not observed in the ring phase, but as a result of changing to mNG, fluorescence was observed even in the ring phase. In addition, an ADAR fusion gene expression strain was created for TZF at the gametocyte stage, and completed preparations for target RNA analysis of TZF.

Project details

Basic Science

Apr 2021 — Mar 2024

$35,721

Japan