Time to switch gears: how long noncoding RNAs function as epigenetic regulators in Apicomplexan parasites.

Long noncoding RNAs (lncRNA) are emerging as important regulators of gene expression in eukaryotes. In recent years, a large repertoire of lncRNA were discovered in Apicomplexan parasites and were implicated in several mechanisms of gene expression, including marking genes for activation, contributing to the formation of subnuclear compartments and organization, regulating the deposition of epigenetic modifications, influencing chromatin and chromosomal structure and manipulating host gene expression. Here, we aim to update recent knowledge on the role of lncRNAs as regulators in Apicomplexan parasites and highlight the possible molecular mechanisms by which they function. We hope that some of the hypotheses raised here will contribute to further investigation and lead to new mechanistic insight and better understanding of the role of lncRNA in parasite's biology.

Beyond splicing: serine-arginine proteins as emerging multifaceted regulators of RNA metabolism in malaria parasites.

The serine-arginine-rich (SR) proteins play an exceptionally important role in eukaryotic gene expression, primarily by regulating constitutive and alternative splicing events. In addition to their primary role as splicing factors, SR proteins have emerged as multifunctional RNA-binding proteins that act as key regulators of almost every step of RNA metabolism. As in higher eukaryotes, Plasmodium parasites encode several SR proteins, which were implicated in pre-mRNA splicing. However, only a few have been characterized and their biological roles remain understudied. Intriguingly, in addition to splicing regulation, unexpected functions of particular SR proteins have been reported in Plasmodium in recent years. Here, we highlight the key characteristics and different noncanonical splicing functions of SR proteins and discuss potential mechanisms, which might be involved in their multifaceted functionality in Plasmodium.

A nuclear redox sensor modulates gene activation and var switching in Plasmodium falciparum.

The virulence of Plasmodium falciparum, which causes the deadliest form of human malaria, is attributed to its ability to evade the human immune response. These parasites "choose" to express a single variant from a repertoire of surface antigens called PfEMP1, which are placed on the surface of the infected red cell. Immune evasion is achieved by switches in expression between var genes, each encoding a different PfEMP1 variant. While the mechanisms that regulate mutually exclusive expression of var genes are still elusive, antisense long-noncoding RNAs (lncRNAs) transcribed from the intron of the active var gene were implicated in the "choice" of the single active var gene. Here, we show that this lncRNA colocalizes with the site of var mRNA transcription and is anchored to the var locus via DNA:RNA interactions. We define the var lncRNA interactome and identify a redox sensor, P. falciparum thioredoxin peroxidase I (PfTPx-1), as one of the proteins associated with the var antisense lncRNA. We show that PfTPx-1 localizes to a nuclear subcompartment associated with active transcription on the nuclear periphery, in ring-stage parasite, when var transcription occurs. In addition, PfTPx-1 colocalizes with S-adenosylmethionine synthetase (PfSAMS) in the nucleus, and its overexpression leads to activation of var2csa, similar to overexpression of PfSAMS. Furthermore, we show that PfTPx-1 knockdown alters the var switch rate as well as activation of additional gene subsets. Taken together, our data indicate that nuclear PfTPx-1 plays a role in gene activation possibly by providing a redox-controlled nuclear microenvironment ideal for active transcription.

Emerging biology of noncoding RNAs in malaria parasites.

In eukaryotic organisms, noncoding RNAs (ncRNAs) have been implicated as important regulators of multifaceted biological processes, including transcriptional, posttranscriptional, and epigenetic regulation of gene expression. In recent years, it is becoming clear that protozoan parasites encode diverse ncRNA transcripts; however, little is known about their cellular functions. Recent advances in high-throughput "omic" studies identified many novel long ncRNAs (lncRNAs) in apicomplexan parasites, some of which undergo splicing, polyadenylation, and encode small proteins. To date, only a few of them are characterized, leaving a big gap in our understanding regarding their origin, mode of action, and functions in parasite biology. In this review, we focus on lncRNAs of the human malaria parasite Plasmodium falciparum and highlight their cellular functions and possible mechanisms of action.

Neutrophils impose strong immune pressure against PfEMP1 variants implicated in cerebral malaria.

Plasmodium falciparum, the deadliest form of human malaria, remains one of the major threats to human health in endemic regions. Its virulence is attributed to its ability to modify infected red blood cells (iRBC) to adhere to endothelial receptors by placing variable antigens known as PfEMP1 on the iRBC surface. PfEMP1 expression determines the cytoadhesive properties of the iRBCs and is implicated in severe malaria. To evade antibody-mediated responses, the parasite undergoes continuous switches of expression between different PfEMP1 variants. Recently, it became clear that in addition to antibody-mediated responses, PfEMP1 triggers innate immune responses; however, the role of neutrophils, the most abundant white blood cells in the human circulation, in malaria remains elusive. Here, we show that neutrophils recognize and kill blood-stage P. falciparum isolates. We identify neutrophil ICAM-1 and specific PfEMP1 implicated in cerebral malaria as the key molecules involved in this killing. Our data provide mechanistic insight into the interactions between neutrophils and iRBCs and demonstrate the important influence of PfEMP1 on the selective innate response to cerebral malaria.

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.

Transdermal delivery of artemisinins for treatment of pre-clinical cerebral malaria.

Transdermal drug delivery avoids complications related to oral or parenteral delivery - the need for sterility, contamination, gastrointestinal side effects, patient unconsciousness or nausea and compliance. For malaria treatment, we demonstrate successful novel transdermal delivery of artemisone (ART) and artesunate. The incorporation of ART into a microemulsion (ME) overcomes the limitations of the lipophilic drug and provides high transcutaneous bioavailability. ART delivery to the blood (above 500 ng/ml) was proved by examining the sera from treated mice, using a bioassay in cultured Plasmodium falciparum. Skin spraying of ART-ME eliminated P. berghei ANKA in an infected mouse model of cerebral malaria (CM) and prevented CM, even after a late treatment with a relatively small amount of ART (13.3 mg/kg). For comparison, the artesunate (the most used commercial artemisinin) formulation was prepared as ART. However, ART-ME was about three times more efficient than artesunate-ME. The solubility and stability of ART in the ME, taken together with the successful transdermal delivery leading to animal recovery, suggest this formulation as a potential candidate for transdermal treatment of malaria.