Plasmodium falciparum, the causative agent of the most virulent form of human malaria, is responsible for between one and two million deaths per year. Since the recent reemergence P. falciparum is in large part a result of increased drug resistance, understanding of drug resistance mechanisms and identification of novel drug targets are urgent goals. Of fundamental importance to both of these goals is an increased knowledge of the mechanisms regulating gene expression in P. falciparum. Several observations indicate that posttranslational regulation of gene expression plays a role at multiple levels in this organism, including mRNA splicing, polyadenylation, cleavage of polycistronic precursor mRNAs, and mRNA stability. However, little is known about the details of these processes in P. falciparum. RNA binding proteins have been shown to be involved in all of these processes in other organisms. This proposal is designed to identify RNA binding proteins in P. falciparum and to begin to assess their functions in this organism. We will use a multi-faceted approach to isolate RNA binding proteins and their genes. This approach will include PCR amplification using primers corresponding to conserved RNA binding domains, screening of cDNA expression libraries with both single- and double-stranded RNA probes, and direct biochemical purification of abundant RNA binding proteins by affinity chromatography. Full length cDNA sequences will then be obtained by 5' and 3' RACE or by screening cDNA libraries. Once clones encoding RNA binding proteins are in hand, the proteins will be expressed and antibodies produced. Developmental regulation and subcellular localization will be assessed to provide information regarding the proteins' functions. General RNA binding preferences will be determined in competition experiments. Specific cellular RNA targets will be identified using an immunoprecipitation approach and/or by iterative selection from a random RNA library (SELEX). These experiments will identify the first RNA binding proteins from P. falciparum, and provide evidence regarding the function of the proteins in the regulation of P. falciparum gene expression. The information obtained regarding gene expression may prove relevant to our understanding of drug resistance mechanisms. Moreover, since genetic mechanisms in lower eukaryotes are frequently divergent, novel proteins identified during this study could provide new targets for chemotherapeutic intervention in malaria.
