Genome-wide analysis in Plasmodium falciparum reveals early and late phases of RNA polymerase II occupancy during the infectious cycle

Abstract

BackgroundOver the course of its intraerythrocytic developmental cycle (IDC), the malaria parasite Plasmodium falciparum tightly orchestrates the rise and fall of transcript levels for hundreds of genes. Considerable debate has focused on the relative importance of transcriptional versus post-transcriptional processes in the regulation of transcript levels. Enzymatically active forms of RNAPII in other organisms have been associated with phosphorylation on the serines at positions 2 and 5 of the heptad repeats within the C-terminal domain (CTD) of RNAPII. We reasoned that insight into the contribution of transcriptional mechanisms to gene expression in P. falciparum could be obtained by comparing the presence of enzymatically active forms of RNAPII at multiple genes with the abundance of their associated transcripts.ResultsWe exploited the phosphorylation state of the CTD to detect enzymatically active forms of RNAPII at most P. falciparum genes across the IDC. We raised highly specific monoclonal antibodies against three forms of the parasite CTD, namely unphosphorylated, Ser5-P and Ser2/5-P, and used these in ChIP-on-chip type experiments to map the genome-wide occupancy of RNAPII. Our data reveal that the IDC is divided into early and late phases of RNAPII occupancy evident from simple bi-phasic RNAPII binding profiles. By comparison to mRNA abundance, we identified sub-sets of genes with high occupancy by enzymatically active forms of RNAPII and relatively low transcript levels and vice versa. We further show that the presence of active and repressive histone modifications correlates with RNAPII occupancy over the IDC.ConclusionsThe simple early/late occupancy by RNAPII cannot account for the complex dynamics of mRNA accumulation over the IDC, suggesting a major role for mechanisms acting downstream of RNAPII occupancy in the control of gene expression in this parasite.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-959) contains supplementary material, which is available to authorized users.

Antagonistic roles of NOT1 paralogues in the timing of gene expression inPlasmodium falciparum

“…To study this, we explored the effect of the PfNot1.1 deletion on both transcription and mRNA decay. To study transcription, we assessed the recruitment of the elongating form of RNAPII at six time points across the IDC by chromatin-immunoprecipitation using Ser2/5-P antibody raised previously [10]. Transcriptional status of RNAPII is known to be associated with sequential changes of phosphorylation at the C-terminal domain (CTD) of RPB1, with phosphorylation of Ser5 alone during transcriptional initiation and phosphorylation of both Ser2 and Ser5 (Ser2/5-P) when the polymerase is engaged in elongation [56].…”

“…These 27 ApiAP2 proteins display distinct expression profiles covering the entire IDC [8] and their recognition motifs are located in the promoter regions of most genes whose expression profiles are positively correlated to expression of the corresponding ApiAP2 gene [9]. In addition to transcriptional initiation, there is mounting evidence that multiple processes of post-transcriptional regulation are also critical for regulation of mRNA abundance through the P. falciparum IDC [10–12]. This is supported by the abundant presence RNA-binding proteins [13, 14] and RNA degradation components [15] in the P. falciparum genome.…”

Plasmodium Falciparum Regulates Transcriptional Output by Modulating The Activity of RNA Polymerase II Rather Than Its Recruitment

“…We have successfully raised and used highly speci c monoclonal antibodies to detect the unphosphorylated heptad repeat, the monophosphorylated Ser5-P form and the diphosphorylated (Ser2/5-P) form of RNAPII in P falciparum, demonstrating that the heptad motifs are modi ed in the parasite as they are in other organisms. While there has been discussion as to the possible divergence of function of CTD phosphorylation in the parasite ( [18][19][20] and reviewed in Rai 2014 [21], the simplest understanding is that the different forms are associated with the same enzymatic states of RNAPII seen in other model systems. Thus, the unphosphorylated, Ser5-P and Ser2/5-P heptad repeats would mark the pre-initiating, initiating, and elongating forms of the polymerase, respectively.…”

“…In our previous study, we used these phosphoisoform-speci c monoclonal antibodies to map the differential association of RNAPII to the entire parasite genome at six 8-hour time intervals across the IDC via chromatin immunoprecipitation (ChIP) followed by the probing of pan-genomic microarrays (ChIPchip) [21]. We made the striking observation that most genes differentially expressed across the IDC are starkly divided in the timing of RNAPII occupancy [21]. While there is a large group of genes bound by RNAPII early in the IDC, a complementary set of genes is bound by RNAPII in the late stage.…”

Real-time in vivo Global Transcriptional Dynamics During Plasmodium falciparum Blood-stage Development

“…Since the advent of the complete P. falciparum genome sequence, numerous studies have utilized whole-genome methods to measure transcript abundance from populations of parasite-infected red blood cells [11][12][13]31,52 as well as, more recently, single cells 53,54 . In addition, other studies have measured transcriptional activity 38,55 , mRNA half-lives 56 , RNA Pol II binding 57 and transcription start sites 58 throughout development.…”