Patterns of Gene-Specific and Total Transcriptional Activity during thePlasmodium falciparumIntraerythrocytic Developmental Cycle

Abstract

The relationships among gene regulatory mechanisms in the malaria parasite Plasmodium falciparum throughout its asexual intraerythrocytic developmental cycle (IDC) remain poorly understood. To investigate the level and nature of transcriptional activity and its role in controlling gene expression during the IDC, we performed nuclear run-on on whole-transcriptome samples from time points throughout the IDC and found a peak in RNA polymerase II-dependent transcriptional activity related to both the number of nuclei per parasite and variable transcriptional activity per nucleus over time. These differential total transcriptional activity levels allowed the calculation of the absolute transcriptional activities of individual genes from gene-specific nuclear run-on hybridization data. For half of the genes analyzed, sense-strand transcriptional activity peaked at the same time point as total activity. The antisense strands of several genes were substantially transcribed. Comparison of the transcriptional activity of the sense strand of each gene to its steady-state RNA abundance across the time points assayed revealed both correlations and discrepancies, implying transcriptional and posttranscriptional regulation, respectively. Our results demonstrate that such comparisons can effectively indicate gene regulatory mechanisms in P. falciparum and suggest that genes with diverse transcriptional activity levels and patterns combine to produce total transcriptional activity levels tied to parasite development during the IDC.Plasmodium falciparum, the most deadly human malaria parasite, undergoes many developmental changes and thrives in both human and mosquito hosts. Since the genome sequence of P. falciparum became available (17), microarray studies of its intraerythrocytic developmental cycle (IDC) (3, 37) and other asexual and bloodborne stages (34) have demonstrated that differential gene expression is integral to the parasite's development. The stage specificity of steady-state RNA expression supports previous observations that the morphological changes of the IDC correspond to many less-apparent changes in physiology, such as protein and nucleic acid metabolism (10, 19) and sensitivity to small molecules (14, 22, 61). The Plasmodium repertoire of gene regulatory mechanisms is comparable to those of other eukaryotes (7, 23), implying that stage-specific gene expression may be regulated at many levels, including chromatin structure, transcriptional activation, and posttranscriptional modulation of translation and stability (11,39,41,59). However, the relationship between such mechanisms during the IDC, specifically whether regulation of primary transcriptional activity determines the steadystate RNA level, remains uncertain for the vast majority of genes.P. falciparum's steady-state expression patterns during the IDC have been hypothesized to result from cis-acting regulatory DNA sequences (e.g., promoters and enhancers), given its canonical basal transcriptional machinery (4,5,55). Numerous studies of individual puta...

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