An SR protein is essential for activating DNA repair in malaria parasites.

Plasmodium falciparum, the parasite responsible for the deadliest form of human malaria, replicates within the erythrocytes of its host, where it encounters numerous pressures that cause extensive DNA damage, which must be repaired efficiently to ensure parasite survival. Malaria parasites, which have lost the non-homologous end joining (NHEJ) pathway for repairing DNA double-strand breaks, have evolved unique mechanisms that enable them to robustly maintain genome integrity under such harsh conditions. However, the nature of these adaptations is unknown. We show that a highly conserved RNA splicing factor, P. falciparum (Pf)SR1, plays an unexpected and crucial role in DNA repair in malaria parasites. Using an inducible and reversible system to manipulate PfSR1 expression, we demonstrate that this protein is recruited to foci of DNA damage. Although loss of PfSR1 does not impair parasite viability, the protein is essential for their recovery from DNA-damaging agents or exposure to artemisinin, the first-line antimalarial drug, demonstrating its necessity for DNA repair. These findings provide key insights into the evolution of DNA repair pathways in malaria parasites as well as the ability of the parasite to recover from antimalarial treatment.

PfSR1 controls alternative splicing and steady-state RNA levels in Plasmodium falciparum through preferential recognition of specific RNA motifs.

Plasmodium species have evolved complex biology to adapt to different hosts and changing environments throughout their life cycle. Remarkably, these adaptations are achieved by a relatively small genome. One way by which the parasite expands its proteome is through alternative splicing (AS). We recently identified PfSR1 as a bona fide Ser/Arg-rich (SR) protein that shuttles between the nucleus and cytoplasm and regulates AS in Plasmodium falciparum. Here we show that PfSR1 is localized adjacent to the Nuclear Pore Complex (NPC) clusters in the nucleus of early stage parasites. To identify the endogenous RNA targets of PfSR1, we adapted an inducible overexpression system for tagged PfSR1 and performed RNA immunoprecipitation followed by microarray analysis (RIP-chip) to recover and identify the endogenous RNA targets that bind PfSR1. Bioinformatic analysis of these RNAs revealed common sequence motifs potentially recognized by PfSR1. RNA-EMSAs show that PfSR1 preferentially binds RNA molecules containing these motifs. Interestingly, we find that PfSR1 not only regulates AS but also the steady-state levels of mRNAs containing these motifs in vivo.

Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum.

The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.

A novel Plasmodium falciparum SR protein is an alternative splicing factor required for the parasites' proliferation in human erythrocytes.

Malaria parasites have a complex life cycle, during which they undergo significant biological changes to adapt to different hosts and changing environments. Plasmodium falciparum, the species responsible for the deadliest form of human malaria, maintains this complex life cycle with a relatively small number of genes. Alternative splicing (AS) is an important post-transcriptional mechanisms that enables eukaryotic organisms to expand their protein repertoire out of relatively small number of genes. SR proteins are major regulators of AS in higher eukaryotes. Nevertheless, the regulation of splicing as well as the AS machinery in Plasmodium spp. are still elusive. Here, we show that PfSR1, a putative P. falciparum SR protein, can mediate RNA splicing in vitro. In addition, we show that PfSR1 functions as an AS factor in mini-gene in vivo systems similar to the mammalian SR protein SRSF1. Expression of PfSR1-myc in P. falciparum shows distinct patterns of cellular localization during intra erythrocytic development. Furthermore, we determine that the predicted RS domain of PfSR1 is essential for its localization to the nucleus. Finally, we demonstrate that proper regulation of pfsr1 is required for parasite proliferation in human RBCs and over-expression of pfsr1 influences AS activity of P. falciparum genes in vivo.

Live imaging of mitosomes and hydrogenosomes by HaloTag technology.

Hydrogenosomes and mitosomes represent remarkable mitochondrial adaptations in the anaerobic parasitic protists such as Trichomonas vaginalis and Giardia intestinalis, respectively. In order to provide a tool to study these organelles in the live cells, the HaloTag was fused to G. intestinalis IscU and T. vaginalis frataxin and expressed in the mitosomes and hydrogenosomes, respectively. The incubation of the parasites with the fluorescent Halo-ligand resulted in highly specific organellar labeling, allowing live imaging of the organelles. With the array of available ligands the HaloTag technology offers a new tool to study the dynamics of mitochondria-related compartments as well as other cellular components in these intriguing unicellular eukaryotes.

High resolution 3D perspective of Plasmodium biology: advancing into a new era.

Apicomplexan parasites exhibit a great variety of complex life cycles that require adaptation to different niches of parasitism. They invade different host cells and highjack their biological functions. Plasmodium falciparum, responsible for the deadliest form of human malaria, causes disease while completely remodeling the erythrocytes of its human host through mechanisms that are only partly understood. Recent developments in ultrastructural technologies offer new opportunities to investigate fundamental aspects in the biology of the parasite in a three-dimensional (3D) perspective. Here we bring together recent work on host cell invasion, hemoglobin uptake, protein export and nuclear dynamics. A comprehensive 3D view of the ultrastructural biology of the parasite may shed new light on cellular mechanisms that underlie the pathogenicity of P. falciparum.