The variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on the surface of P. falciparum infected Red Blood Cells (iRBCs) is a critical virulence factor for malaria1. Each parasite encodes 60 antigenically distinct var genes encoding PfEMP1s, but during infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune evasion mechanism to avoid the host’s antibody responses2,3. The mechanism by which 59 of the 60 var genes are silenced remains largely unknown4–7. Here we show that knocking out the P. falciparum variant-silencing SET gene (PfSETvs), which encodes an ortholog of Drosophila melanogaster ASH1 and controls histone H3 lysine 36 trimethylation (H3K36me3) on var genes, results in the transcription of virtually all var genes in the single parasite nuclei and their expression as proteins on the surface of individual iRBCs. PfSETvs-dependent H3K36me3 is present along the entire gene body including the transcription start site (TSS) to silence var genes. With low occupancy of PfSETvs at both the TSS of var genes and the intronic promoter, expression of var genes coincides with transcription of their corresponding antisense long non-coding RNA (lncRNA). These results uncover a novel role of the PfSETvs-dependent H3K36me3 in silencing var genes in P. falciparum that might provide a general mechanism by which orthologs of PfSETvs repress gene expression in other eukaryotes. PfSETvs knockout parasites expressing all PfEMP1s may also be applied to the development of a malaria vaccine.

BackgroundSchistosomiasis japonica remains a major public health problem in China. Its pathogen, Schistosoma japonicum has a complex life cycle and a unique repertoire of genes expressed at different life cycle stages. Exploring schistosome gene regulation will yield the best prospects for new drug targets and vaccine candidates. MicroRNAs (miRNAs) are a highly conserved class of noncoding RNA that control many biological processes by sequence-specific inhibition of gene expression. Although a large number of miRNAs have been identified from plants to mammals, it remains no experimental proof whether schistosome exist miRNAs.Methodology and ResultsWe have identified novel miRNAs from Schistosoma japonicum by cloning and sequencing a small (18–26 nt) RNA cDNA library from the adult worms. Five novel miRNAs were identified from 227 cloned RNA sequences and verified by Northern blot. Alignments of the miRNAs with corresponding family members indicated that four of them belong to a metazoan miRNA family: let-7, miR-71, bantam and miR-125. The fifth potentially new (non conserved) miRNA appears to belong to a previously undescribed family in the genus Schistosome. The novel miRNAs were designated as sja-let-7, sja-miR-71, sja-bantam, sja-miR-125 and sja-miR-new1, respectively. Expression of sja-let-7, sja-miR-71 and sja-bantam were analyzed in six stages of the life cycle, i.e. egg, miracidium, sporocyst, cercaria, schistosomulum, and adult worm, by a modified stem-loop reverse transcribed polymerase chain reaction (RT-PCR) method developed in our laboratory. The expression patterns of these miRNAs were highly stage-specific. In particular, sja-miR-71 and sja-bantam expression reach their peaks in the cercaria stage and then drop quickly to the nadirs in the schistosomulum stage, following penetration of cercaria into a mammalian host.ConclusionsAuthentic miRNAs were identified for the first time in S. japonicum, including a new schistosome family member. The different expression patterns of the novel miRNAs over the life stages of S. japonicum suggest that they may mediate important roles in Schistosome growth and development.

Antigenic variation of the Plasmodium falciparum multicopy var gene family enables parasite evasion of immune destruction by host antibodies. Expression of a particular var subgroup, termed upsA, is linked to the obstruction of blood vessels in the brain and to the pathogenesis of human cerebral malaria. The mechanism determining upsA activation remains unknown. Here we show that an entirely new type of gene silencing mechanism involving an exonuclease-mediated degradation of nascent RNA controls the silencing of genes linked to severe malaria. We identify a novel chromatin-associated exoribonuclease, termed PfRNase II, that controls the silencing of upsA var genes by marking their transcription start site and intron-promoter regions leading to short-lived cryptic RNA. Parasites carrying a deficient PfRNase II gene produce full-length upsA var transcripts and intron-derived antisense long non-coding RNA. The presence of stable upsA var transcripts overcomes monoallelic expression, resulting in the simultaneous expression of both upsA and upsC type PfEMP1 proteins on the surface of individual infected red blood cells. In addition, we observe an inverse relationship between transcript levels of PfRNase II and upsA-type var genes in parasites from severe malaria patients, implying a crucial role of PfRNase II in severe malaria. Our results uncover a previously unknown type of post-transcriptional gene silencing mechanism in malaria parasites with repercussions for other organisms. Additionally, the identification of RNase II as a parasite protein controlling the expression of virulence genes involved in pathogenesis in patients with severe malaria may provide new strategies for reducing malaria mortality.

Many microbial pathogens, including the malaria parasite Plasmodium falciparum, vary surface protein expression to evade host immune responses. P. falciparium antigenic variation is linked to var gene family-encoded clonally variant surface protein expression. Mututally exclusive var gene expression is partially controlled by spatial positioning; silent genes are retained at distinct perinuclear sites and relocated to transcriptionally active locations for monoallelic expression. We show that var introns can control this process and that var intron addition relocalizes episomes from a random to a perinuclear position. This var intron-regulated nuclear tethering and repositioning is linked to an 18 bp nuclear protein-binding element that recruits an actin protein complex. Pharmacologically induced F-actin formation, which is restricted to the nuclear periphery, repositions intron-carrying episomes and var genes and disrupts mutually exclusive var gene expression. Thus, actin polymerization relocates var genes from a repressive to an active perinuclear compartment, which is crucial for P. falciparium phenotypic variation and pathogenesis.

Background: The transcriptional regulation of Plasmodium during its complex life cycle requires sequential activation and/or repression of different genetic programmes. MicroRNAs (miRNAs) are a highly conserved class of non-coding RNAs that are important in regulating diverse cellular functions by sequence-specific inhibition of gene expression. What is know about double-stranded RNA-mediated gene silencing (RNAi) and posttranscriptional gene silencing (PTGS) in Plasmodium parasites entice us to speculate whether miRNAs can also function in Plasmodium-infected RBCs.

Gametocytogenesis, the process by which malaria parasites produce sexual forms that can infect mosquitoes, is essential for the transmission of malaria. A transcriptional switch of the pfap2-g gene triggers sexual commitment, but how the complex multi-step process is precisely programed remains largely unknown. Here, by systematic functional screening of a panel of ApiAP2 transcription factors, we identify six new ApiAP2 members associated with gametocytogenesis in Plasmodium falciparum. Among these, PfAP2-G5 (PF3D7_1139300) was found to be indispensable for gametocytogenesis. This factor suppresses the transcriptional activity of the pfap2-g gene via binding to both the upstream region and exonic gene body, the latter is linked to the maintenance of local heterochromatin structure, thereby preventing initiation of sexual commitment. Removal of this repressive effect through pfap2-g5 knockout disrupts the asexual replication cycle and promotes sexual commitment accompanied by upregulation of pfap2-g expression. However, the gametocytes produced fail to mature fully. Further analyses show that PfAP2-G5 is essential for gametocyte maturation, and causes the down-regulation of pfap2-g and a set of early gametocyte genes activated by PfAP2-G prior to gametocyte development. Collectively, our findings reveal a regulation cascade of gametocyte production in malaria parasites, and provide a new target for transmission blocking interventions.

Dynamic changes in gene positioning contribute to differential expression of virulence-related gene families in protozoan pathogens; however, the role of nuclear architecture in gene expression in the human malaria parasite Plasmodium falciparum remains poorly understood. Here we investigated the developmentally regulated ribosomal RNA (rRNA) gene family in P. falciparum, which, unlike that in most eukaryotes, contains only a few unlinked copies of rRNA genes scattered over the subtelomeric regions of several chromosomes. We show that active and silent members of this gene family cluster in a single perinuclear nucleolus. This rDNA nuclear confinement is DNA sequence dependent, as plasmids carrying rDNA fragments are targeted to the nucleolus. Likewise, insertion of an rDNA sequence into a subtelomere from a chromosome lacking rRNA genes leads to repositioning in the nucleolus. Furthermore, we observed that rDNA spatial organization restricted interchromosomal interactions, as chromosome end-bearing rRNA genes were found to be preferentially juxtaposed, demonstrating nonrandom association of telomeres. Using Br-UTP incorporation, we observed two α-amanitin-resistant nucleolar transcription sites that disappeared when the rDNA cluster broke up in the replicative blood stages. Taken together, our results provide conceptual insights into functionally differentiated nuclear territories and their role in gene expression in malaria parasites.nucleus | nucleolus | telomeres | transcription regulation E ukaryotic nuclei are highly organized structures in which DNA and proteins are confined to specific subcompartments such as nucleoli, nuclear bodies, nuclear speckles, transcription and replication foci, and chromosome territories. The most prominent intranuclear domain is the nucleolus, the ribosome factory. This subcompartment is formed upon transcription of the ribosomal DNA (rDNA) by RNA polymerase I (Pol I). The formation of single or multiple nucleoli is seemingly linked to the genomic organization of the tandemly repeated rDNA. For example, budding yeast has ∼150 rDNA units clustered on a single chromosome (chromosome 12) and forms one nucleolus, whereas human cells have ∼400 tandem repeats clustered on five chromosomes (chromosomes 13, 14, 15, 21, and 22) and form multiple nucleoli per nucleus (1, 2). In addition, in yeast, the deletion of chromosomal rDNA and replacement by an equivalent number of plasmids bearing single rDNA repeats generates multiple small nucleoli (3), highlighting the importance of the tandem arrangement in nucleolar organization.In the human malaria parasite Plasmodium falciparum, rDNA is not arranged in tandem arrays; instead, single rDNA units (18S-5.8S-28S) are spread out on different chromosomes (chromosomes 1, 5, 7, 11, and 13, Fig. 1A) (4-6). Moreover, the few rDNA units are slightly different in sequence and are differentially transcribed over the course of the parasite developmental cycle (7-9). In this work, we investigated the nuclear position of active and silent members of this ...

Heterochromatin-associated gene silencing controls multiple physiological processes in malaria parasites, however, little is known concerning the regulatory network and cis-acting sequences involved in the organization of heterochromatin and how they modulate heterochromatic gene expression. Based on systematic profiling of genome-wide occupancy of eighteen Apicomplexan AP2 transcription factors by ChIP-seq analysis, we identify and characterize eight heterochromatin-associated factors (PfAP2-HFs), which exhibit preferential enrichment within heterochromatic regions but with differential coverage profiles. Although these ApiAP2s target euchromatic gene loci via specific DNA motifs, they are likely integral components of heterochromatin independent of DNA motif recognition. Systematic knockout screenings of ApiAP2 factors coupled with RNA-seq transcriptomic profiling revealed three activators and three repressors of heterochromatic gene expression including four PfAP2-HFs. Notably, expression of virulence genes is either completely silenced or significantly reduced upon the depletion of PfAP2-HC. Integrated multi-omics analyses reveal autoregulation and feed-forward loops to be common features of the ApiAP2 regulatory network, in addition to the occurrence of dynamic interplay between local chromatin structure and ApiAP2s in transcriptional control. Collectively, this study provides a valuable resource describing the genome-wide landscape of the ApiAP2 family and insights into functional divergence and cooperation within this family during the blood-stage development of malaria parasites.