Project description:The quality control and export of mRNA by RNA-binding proteins are necessary for the survival of malaria parasites, which have complex life cycles. Malarial nuclear poly(A) binding protein 2 (NAB2), ALWAYS EARLY (ALY) and serine/arginine-rich (SR) proteins, such as nucleolar protein 3 (NPL3), G-strand binding protein 2 (GBP2) and SR1, are involved in nuclear mRNA export in malaria parasites. However, their functions and cellular localization are not fully understood. In this study, we found that NAB2 and SR1, but not ALY, NPL3 or GBP2, played essential roles in the asexual development of malaria parasites, while GBP2 was involved in gametocyte production. Moreover, GBP2 localized to both the nucleus and cytoplasm of malaria parasites and interacted with the proteins ALBA4, DOZI and CITH, which play roles in translational repression. Our findings suggest that malarial GBP2 may be involved in the recruitment of ALBA4, DOZI and CITH. Immunoprecipitation coupled to mass spectrometry (IP-MS) revealed that PHAX domain-containing protein, an adaptor protein for exportin-1, also interacted with GBP2, suggesting that mRNA export occurs via the PHAX domain-containing protein pathway in malaria parasites. Fluorescence live cell imaging revealed that malarial NAB2 localized at the nuclear periphery and co-localized with NUP205. Moreover, using IP-MS, we found that malarial NAB2 interacted with transportin. RNA immunoprecipitation coupled to RNA sequencing revealed that malarial NAB2 bound directly to 143 mRNAs, including those encoding 40S and 60S ribosomal proteins. This indicates that malarial NAB2 is involved in general mRNA assembly and is shuttled between the nucleus and cytoplasm. Our findings suggest that unique mRNA export and post-transcriptional gene regulation mediated by RNA-binding proteins occur in malaria parasites.
Project description:The purpose of this research is to identify and evaluate the global gene expression of the rodent malaria parasites Plasmodium yoelii, Plasmodium berghei and Plasmodium chabaudi blood-stage parasites and specifically compare the blood stage gene expression profiles of samples derived from previous studies on Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi
Project description:During the malaria infection, Plasmodium parasites invade the host’s red blood cells where they can differentiate into two different life forms. The majority will replicate asexually and infect new erythrocytes. A small percentage, however, will transform into gametocytes – a specialized sexual stage able to survive and develop when taken up by Anopheles mosquito. As the gametocytes ensure the parasite’s transmission to a new host, their generation is an attractive target for new antimalarial interventions. The molecular mechanisms controlling gametocytogenesis, however, remain largely unknown due to the technical challenges: the early gametocytes are morphologically indistinguishable from asexual parasites and present in very low numbers during the infection. Recently, AP2-G - a transcription factor from an apicomplexa-specific apiAP2 family – was described as indispensable for gametocyte commitment in both human malaria parasite Plasmodium falciparum and rodent malaria model Plasmodium berghei. Therefore, we have decided to test whether the overexpression of this factor alone could increase gametocyte production and enable the investigation of uncharacterised, earliest stages of gametocyte development. To this end, we have engineered PBGAMi - a Plasmodium berghei line, in which all parasites were ap2-g deficient by default but able to overexpress it when induced with rapamycin. While the control parasites (PBGAMi R-), as expected, differentiated into asexual forms (schizonts) only, almost all rapamycin-treated parasites (PBGAMi R+) transformed into gametocytes. We used the generated line to perform RNA-seq analysis of the R- and R+ populations at different time points of their development and identify the changes arising between them, mapping the sequence of events leading to the formation of gametocytes.
Project description:During the malaria infection, Plasmodium parasites invade the host’s red blood cells where they can differentiate into two different life forms. The majority will replicate asexually and infect new erythrocytes. A small percentage, however, will transform into gametocytes – a specialized sexual stage able to survive and develop when taken up by Anopheles mosquito. As the gametocytes ensure the parasite’s transmission to a new host, their generation is an attractive target for new antimalarial interventions. The molecular mechanisms controlling gametocytogenesis, however, remain largely unknown due to the technical challenges: the early gametocytes are morphologically indistinguishable from asexual parasites and present in very low numbers during the infection. Recently, AP2-G - a transcription factor from an apicomplexa-specific apiAP2 family – was described as indispensable for gametocyte commitment in both human malaria parasite Plasmodium falciparum and rodent malaria model Plasmodium berghei. Therefore, we have decided to test whether the overexpression of this factor alone could increase gametocyte production and enable the investigation of uncharacterised, earliest stages of gametocyte development. To this end, we have engineered PBGAMi - a Plasmodium berghei line, in which all parasites were ap2-g deficient by default but able to overexpress it when induced with rapamycin. While the control parasites (PBGAMi R-), as expected, differentiated into asexual forms (schizonts) only, almost all rapamycin-treated parasites (PBGAMi R+) transformed into gametocytes. We used the generated line to perform RNA-seq analysis of the R- and R+ populations at different time points of their development and identify the changes arising between them, mapping the sequence of events leading to the formation of gametocytes. At the same time we have generated purified transcriptomes of male and female gametocytes for the reference