FUNCTIONAL CHARACTERIZATION OF A PUF FAMILY RNA-BINDING PROTEIN DURING SEXUAL DEVELOPMENT OF THE MALARIA PARASITE PLASMODIUM FALCIPARUM

Open Access
Author:
Li, Jinfang
Graduate Program:
Entomology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
November 21, 2005
Committee Members:
  • Liwang Cui, Committee Chair
  • Gary Felton, Committee Member
  • Diana Lynn Cox Foster, Committee Member
  • Paul Lee Babitzke, Committee Member
Keywords:
  • RNA-binding
  • Puf
  • PfPuf1
  • PfPuf2
  • sexual development
Abstract:
Despite tremendous worldwide efforts to control malaria, it is still a major public health problem and a heavy social and economic burden in over 90 countries. Among the four protozoan species that cause malaria in humans, Plasmodium falciparum is the most fatal, accounting for 1.5-2.7 million deaths and 300-500 million clinical cases each year. Drug resistance in Plasmodium and insecticide resistance in vector mosquitoes are probably responsible for the resurgence of the disease in the world. Novel drugs and control measures are desperately needed to bring this disease under control. To this aim, we need to better understand the fundamental biology and the molecular mechanisms governing the complicated developmental cycle of the parasite. The recently completed genome sequence of the P. falciparum is a milestone in malaria research, which has contributed greatly to our understanding of the molecular biology of the parasite, and led to the discovery of parasite-specific metabolic pathways and identification of potential targets for the development of new drugs and vaccines. Meanwhile, deciphering the functions of individual genes remains a formidable task of the post-genomic era. Malaria is caused by the bite of an infected mosquito, which injects Plasmodium sporozoites into the human circulatory system. These sporozoites invade liver cells and undergo multiple rounds of schizogony to produce thousands of merozoites, which are released into the blood to invade erythrocytes. While the majority of the parasites undergo cycles of asexual multiplication in the erythrocytes, which is responsible for the clinical symptoms of the disease, some merozoites initiate sexual development to produce gametocytes. Gametocytes are the only form that initiates further sporogonic development in the mosquitoes, an obligatory process for the transmission of the parasite. The complex life cycle of the malaria parasites is a challenge for vaccine designs, which preferentially target multiple developmental stages. Among them, transmission-blocking vaccine that targets parasite antigens of sexual stages aims at interrupting the transmission cycle of malaria. Although morphological changes during sexual development of the malaria parasite were known almost a century ago and a number of sexual stage-specific genes have been identified and functionally characterized, a comprehensive understanding of the molecular and cellular biology of malaria sexual development is still lacking. During studies examing the sexual development of the malaria parasite, members of a family of RNA-binding proteins were identified in P. falciparum gametocytes by using RNA differential display. This family was named Puf after the two first-characterized proteins Pumilio in Drosophila melanogaster and fem-3 binding factor (FBF) in Caenorhabditis elegans. Puf is an evolutionarily conserved RNA binding protein family in eukaryotes. The eight imperfect tandem repeats in Puf proteins constitute the essential RNA binding domain (RBD), which forms a rainbow structure with the concave surface interacting with the target mRNA. The target mRNAs characterized to date contain conserved elements in their 3¡¯ untranslated regions (UTRs), and binding of Puf to these elements imposes translational repression on the target mRNA. The biological functions of Puf family proteins have been characterized in a few biological systems. Although varying from system to system, it has been postulated that the ancestral function of Puf is to promote proliferation and suppress differentiation. The two Puf members in P. falciparum, PfPuf1 and PfPuf2, are preferentially expressed in gametocytes. The finding that Puf proteins in D. melanogaster and C. elegans have a conserved function in germline stem cell maintenance has prompted us to investigate the functions of PfPuf1 and PfPuf2 during P. falciparum gametocytogenesis. PfPuf1 and PfPuf2 mRNAs are spliced and located on chromosome 5 and 4, respectively. PfPuf1 encodes a much larger protein (1894 amino acids) than PfPuf2 (514 amino acids). Phylogenetic analysis of Pufs showed that the RBDs of PfPuf1 and PfPuf2 only share 27% amino acid identity, with PfPuf1 being more related to Pufs in plants and fungi and PfPuf2 more related to FBF in C. elegans. Reverse transcriptase polymerase chain reaction (RT-PCR), northern blots and western blots confirmed the preferential expression of both Puf genes in gametocytes. Using RNA ligase-mediated rapid amplification of cDNA end and primer extension, the transcription initiation site of PfPuf2 was mapped to 297 bp upstream of the translation start codon. Since the target genes for PfPuf proteins remain to be identified, the Drosophila nanos-responsive element (NRE) sequence in the hunchback 3¡¯ UTR was used as a conserved artificial target sequence to evaluate the RNA-binding activity of PfPufs by using gel mobility shift assays and the yeast three-hybrid system. The results showed that both PfPuf RBDs displayed specific RNA binding activity to the conserved NRE sequence. This result suggests that both Puf members in P. falciparum utilize a similar mechanism of translation control of their target genes. To further explore the functions of PfPuf genes, the newly developed parasite transfection technology was applied to disrupt PfPuf2. A plamid construct was designed to truncate PfPuf2 at the 8th Puf repeat. After electroporation of the ring- stage parasites, pyrimethamine was used to select resistant parasites. Parasites with the construct stably integrated into the PfPuf2 locus were obtained and cloned by limiting dilution. Disruption of PfPuf2 in these single parasite clones was confirmed by integration-specific PCR, genomic Southern blot, and RT-PCR. Phenotypic analysis showed that PfPuf2 disruptions did not affect asexual growth of the parasite, but gametocytes in the disruptants were formed at a much accelerated rate. The disruption of Puf genes provides us an opportunity to identify the Puf target genes through genome-wide microarray analysis.