Antigenic variation in Plasmodium falciparum malaria: The role of the intron promoter in var gene silencing
Final Report Abstract
Antigenic variation of the malaria parasite Plasmodium falciparum is caused by switches in gene expression of the multicopy var gene family. Only one gene is expressed at a time while the remaining copies are maintained in a silent state. The expression of each var gene is regulated by two separate promoters, one upstream of the coding region and a second within the intron. These two promoters appear to be co-regulated, with the upstream promoter active in expressed var genes while the intron promoter transcribes a truncated, “sterile” transcript in silent genes. Although dependence on S-Phase implies that alterations in chromatin structure are involved, the mechanism of var gene silencing remains unknown. The goal of the proposed research activity was to investigate the role of the intron promoter in var gene silencing. Two specific aims were addressed experimentally: In Aim A the effect of var upstream elements on the transcriptional activity of the intron promoter was analyzed. Bidirectional reporter constructs were used to measure the promoter activity of introns in both directions simultaneously. Several introns representing different subgroups of var genes were tested and all were found to contain bidirectional promoters. This finding was corroborated by RNA analysis of malaria parasites by Northern Blot and RT-PCR. In addition to the previously described “sterile” trancripts of exon 2, non-coding antisense RNAs of exon 1 were detected. Nuclear, non-coding RNAs in many organisms are involved in chromatin assembly and epigenetic gene regulation and the non-coding RNAs originating from var introns might play a similar role in var gene regulation. The experiments carried out within this study provide the first experimental evidence for the identification of antisense RNA transcripts of exon 1. Based on previous data, it was expected that presence of var upstream elements would influence the intron promoter. However, upon addition of upstream promoter elements into bidirectional reporter constructs no significant changes of intron promoter activity were observed, neither in transient transfection assays nor in transfected parasites with stably maintained episomes. In Aim B it was sought to establish a tetracycline repressor (TetR) based inducible system to regulate the intron promoter activity and apply it for analyzing its effect on var gene silencing. In a first step, tetracycline operators (tetO) were fused to the intron promoter. Depending on their position, the tetO sequences strongly influenced the promoter activity of the intron. Nevertheless, the intron-tetO fusions were capable of silencing a var upstream promoter, which is an important requirement for the development of an inducible genetic system for var gene regulation. Unfortunately, the progress of this project has been hampered by difficulties in obtaining a tetR-expressing parasite line, but alternative experimental approaches are currently being pursued.
Publications
- Molecular Parasitology Meeting XVI, 11-15. Sept. 2005, Woods Hole, MA, USA: The promoters found in var gene introns of P. falciparum are bidirectional, producing both sense and antisense nuclear noncoding RNAs
Epp C, Dzikowski R, Frank M, Li F, Deitsch KW
- Second Annual BioMalPar Conference on the Biology and Pathology of the Malaria parasite, 5-8. April 2006, EMBL, Heidelberg: Bidirectional promoters found in var gene introns produce both sense and antisense nuclear non-coding RNAs
Epp C, Dzikowski R, Frank M, Li F, Deitsch KW
