Patterns of gene-specific and total transcriptional activity during the Plasmodium falciparum intraerythrocytic 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.

FIG. 1.

Total transcriptional activity across the 48-h IDC. Transcriptional activity was measured in counts per minute (cpm) of total ³²P incorporation by nuclei harvested during four independent time courses. Nuclei were treated with RNAP II inhibitor α-amanitin (+) or an equal volume of water (−) prior to incubation at 37°C for 30 min in the presence of [α-³²P]UTP. x axes represent hours postreinvasion. Time points during time course C occur at 2 h resolution during the final 10 h.

FIG. 2.

Fit of stage-specific model to observed transcriptional activity. (A) Total transcriptional activity per parasite for each stage as calculated by fitting algorithm. The vectors representing the best-fit characteristic transcriptional activity levels of the six morphological stages as output by the algorithm for time courses A and B are graphed. ER, early rings; LR, late rings; ET, early trophozoites; MT, mid-trophozoites; LT, late trophozoites; S, schizonts. (B) Relationship between model's output for transcriptional activity per parasite and number of nuclei per parasite. The number of nuclei per parasite was counted for parasites of each of the six stages (x axis) and compared to the characteristic transcriptional activity level for that stage (from panel A, y axis).

FIG. 3.

Nuclear run-on gene-specific hybridization. (A) Total-incorporation nuclear run-on of time course D is depicted as in Fig. 1, with time points which were assayed by gene-specific nuclear run-on (T12, T33, T39, and T45) shown in black. x axes represent hours postreinvasion. (B) Single-stranded DNA probes were slot blotted onto membranes, which were hybridized with run-on-labeled [³²P]RNA from time points T12, T33, T39, and T45 and imaged by exposure of a phosphorimager screen. The legend describes which RNA strand was detected. rRNA slots were cut from their filters and exposed separately to avoid interference between their signals and those of adjacent slots but are shown inset in their original positions.

FIG. 4.

Ranges of transcriptional activity across time points for sense strands of specific genes. The signal from the sense strand of each gene (shown in the same order as in Table 1) as detected by phosphorimager (see Table S4 in the supplemental material), corrected for single-stranded DNA probe purity and [³²P]U content of the target RNA sequence, was normalized by the total radioactivity applied to its filter during hybridization and then multiplied by the total incorporation for that time point (relative to that of T12), yielding the absolute intensity value for that strand. The mean sense-strand signal for each gene across all four time points (diamonds) is shown between the maximum and minimum values (whiskers) among the four time points.

FIG. 5.

Relationship between transcriptional activity and steady-state RNA levels. The corrected, normalized transcriptional activity of each strand, detected by gene-specific nuclear run-on in the slot blots depicted in Fig. 3 and multiplied by the total transcriptional activity for each time point, is displayed with respect to the maximum value for the gene among the four time points (bars: S, sense; AS, antisense). Levels of steady-state RNA (RNA) assayed by reverse transcription and real-time PCR are graphed with respect to their maximum values among the time points. All y axes represent the fraction of maximum for both transcriptional activity and steady-state RNA. Panels A to F represent the six gene groups assayed, as described in Table 1. Asterisks denote genes for which not all data were available; genes of low mean value were not included (see Table S3 in the supplemental material).