Recent advances in high-throughput sequencing present a new opportunity to deeply probe an organism's transcriptome. In this study, we used Illumina-based massively parallel sequencing to gain new insight into the transcriptome (RNA-Seq) of the human malaria parasite, Plasmodium falciparum. Using data collected at seven time points during the intraerythrocytic developmental cycle, we (i) detect novel gene transcripts; (ii) correct hundreds of gene models; (iii) propose alternative splicing events; and (iv) predict 5′ and 3′ untranslated regions. Approximately 70% of the unique sequencing reads map to previously annotated protein-coding genes. The RNA-Seq results greatly improve existing annotation of the P. falciparum genome with over 10% of gene models modified. Our data confirm 75% of predicted splice sites and identify 202 new splice sites, including 84 previously uncharacterized alternative splicing events. We also discovered 107 novel transcripts and expression of 38 pseudogenes, with many demonstrating differential expression across the developmental time series. Our RNA-Seq results correlate well with DNA microarray analysis performed in parallel on the same samples, and provide improved resolution over the microarray-based method. These data reveal new features of the P. falciparum transcriptional landscape and significantly advance our understanding of the parasite's red blood cell-stage transcriptome.

Summary Intracellular pathogens have devised mechanisms to exploit their host cells to ensure their survival and replication. The malaria parasite Plasmodium falciparum relies on an exchange of metabolites with the host for proliferation. We describe the first mass spectrometry-based metabolomic analysis of the parasite throughout its 48-hour intraerythrocytic developmental cycle. Our results reveal a general modulation of metabolite levels by the parasite, with numerous metabolites varying in phase with the developmental cycle. Others differed from uninfected cells irrespective of the developmental stage. Among these was extracellular arginine, which was specifically converted to ornithine by the parasite. To identify the biochemical basis for this effect, we disrupted the plasmodium arginase gene in the rodent malaria model P. berghei. These parasites were viable but did not convert arginine to ornithine. Our results suggest that systemic arginine depletion by the parasite may be a factor in human malarial hypoargininemia associated with cerebral malaria pathogenesis.

Protein-RNA networks are ubiquitous and central in biological control. We present an approach, termed “RNA Tagging,” that identifies protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or crosslinking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA using high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The method revealed that while RNA-binding proteins productively bind specific RNAs to control their function, they also “sample” RNAs without exerting a regulatory effect. We exploited the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. The RNA Tagging approach is well-suited to detect and analyze protein-RNA networks in vivo.

Proteins bind and control mRNAs, directing their localization, translation and stability. Members of the PUF family of RNA-binding proteins control multiple mRNAs in a single cell, and play key roles in development, stem cell maintenance and memory formation. Here we identified the mRNA targets of a S. cerevisiae PUF protein, Puf5p, by ultraviolet-crosslinking-affinity purification and high-throughput sequencing (HITS-CLIP). The binding sites recognized by Puf5p are diverse, with variable spacer lengths between two specific sequences. Each length of site correlates with a distinct biological function. Crystal structures of Puf5p–RNA complexes reveal that the protein scaffold presents an exceptionally flat and extended interaction surface relative to other PUF proteins. In complexes with RNAs of different lengths, the protein is unchanged. A single PUF protein repeat is sufficient to induce broadening of specificity. Changes in protein architecture, such as alterations in curvature, may lead to evolution of mRNA regulatory networks.

Polyamines are ubiquitous components of all living cells, and their depletion usually causes cytostasis, a strategy employed for treatment of West African trypanosomiasis. To evaluate polyamine depletion as an anti-malarial strategy, cytostasis caused by the co-inhibition of S-adenosylmethionine decarboxylase/ ornithine decarboxylase in Plasmodium falciparum was studied with a comprehensive transcriptome, proteome, and metabolome investigation. Highly synchronized cultures were sampled just before and during cytostasis, and a novel zero time point definition was used to enable interpretation of results in lieu of the developmentally regulated control of gene expression in P. falciparum. Transcriptome analysis revealed the occurrence of a generalized transcriptional arrest just prior to the growth arrest due to polyamine depletion. However, the abundance of 538 transcripts was differentially affected and included three perturbation-specific compensatory transcriptional responses as follows: the increased abundance of the transcripts for lysine decarboxylase and ornithine aminotransferase and the decreased abundance of that for S-adenosylmethionine synthetase. Moreover, the latter two compensatory mechanisms were confirmed on both protein and metabolite levels confirming their biological relevance. In contrast with previous reports, the results provide evidence that P. falciparum responds to alleviate the detrimental effects of polyamine depletion via regulation of its transcriptome and subsequently the proteome and metabolome.Polyamines such as putrescine, spermidine, and spermine are small organic compounds containing two or more amino groups. At physiological pH, these polycations interact electrostatically with numerous anionic macromolecules, thereby stabilizing DNA, RNA, nucleoside triphosphates (e.g. ATP), phospholipids, and proteins (1, 2). These interactions with polyamines can alter DNA conformation, regulate replication and transcription, strengthen membranes, regulate ion channels, and protect DNA and phospholipids from oxidative stress (1-5). Yet polyamines are also implicated in apoptosis (5). Polyamine depletion generally causes cytostasis or growth arrest, which implies that these molecules are involved in cell cycle progression and regulation, and it is speculated that polyamines regulate cyclin degradation (1, 6, 7). Therefore, polyamines are essential for cellular growth, differentiation, and macromolecular synthesis and are ubiquitous components of all living cells, except two orders of Archaea (1). Polyamine metabolism is particularly important in rapidly proliferating cells and has been exploited in the treatment of cancer (1) and parasitic diseases (8). Polyamine metabolism of the malaria parasite Plasmodium falciparum is also a potential target for therapeutic intervention (9, 10).Polyamine and methionine metabolism are closely connected. This is particularly evident in Plasmodium where the two rate-limiting enzymes of polyamine biosynthesis, ornithine decarboxylase (ODC) 2 and S-adenosylme...

SummaryThe genome of the malaria parasite Plasmodium falciparum contains several multicopy gene families, including var, rifin, stevor and Pfmc-2TM. These gene families undergo expression switching and appear to play a role in antigenic variation. It has recently been shown that forcing parasites to express high copy numbers of transcriptionally active, episomal var promoters led to gradual downregulation and eventual silencing of the entire var gene family, suggesting that a limiting titratable factor plays a role in var gene activation. Through similar experiments using rifin, stevor or Pfmc-2TM episomal promoters we show that promoter titration can be used as a general method to downregulate multicopy gene families in P. falciparum. Additionally, we show that promoter titration with var, rifin, stevor or Pfmc-2TM episomal promoters results in downregulation of expression not only of the family to which the episomal promoter belongs, but also members of the other gene families, suggesting that the var-specific titratable factor previously described is shared by all four families. Further, transcriptionally active promoters from different families colocalize within the same subnuclear expression site, indicating that the role that nuclear architecture plays in var gene regulation also likely applies to the other multicopy gene families of P. falciparum.

Alterations in regulatory networks contribute to evolutionary change. Transcriptional networks are reconfigured by changes in the binding specificity of transcription factors and their cognate sites. The evolution of RNA-protein regulatory networks is far less understood. The PUF (Pumilio and FBF) family of RNA regulatory proteins controls the translation, stability, and movements of hundreds of mRNAs in a single species. We probe the evolution of PUF-RNA networks by direct identification of the mRNAs bound to PUF proteins in budding and filamentous fungi and by computational analyses of orthologous RNAs from 62 fungal species. Our findings reveal that PUF proteins gain and lose mRNAs with related and emergent biological functions during evolution. We demonstrate at least two independent rewiring events for PUF3 orthologs, independent but convergent evolution of PUF4/5 binding specificity and the rewiring of the PUF4/5 regulons in different fungal lineages. These findings demonstrate plasticity in RNA regulatory networks and suggest ways in which their rewiring occurs.

Background: PUF protein RNA recognition is critical for target gene regulation. Results: A chemically conserved binding pocket in a subset of PUF proteins recognizes cytosine at different positions upstream of the core PUF recognition sequence. Conclusion: A specialized cytosine-binding pocket introduces qualitative and quantitative differences in RNA recognition by PUF proteins. Significance: Simple adaptations can diversify PUF protein RNA recognition.