IntroductionPlasmodium falciparum is the most damaging malaria pathogen and brings a heavy burden to global health. Host switching and morphological changes in P. falciparum are dependent on an effective gene expression regulatory system. C5 methylation of cytosines is a common RNA modification in eukaryotes, and the NSUN family are essential m5C modification executors. Currently, little is known about this family in Plasmodium spp. In this study, we focus on exploring the function of PfNSUN1 protein.MethodsAn efficient CRISPR/Cas9 gene editing technique was applied to construct the PfNSUN1 knockdown strain. The knockdown efficiency was confirmed by growth curves and western blot experiments. The knockdown transcriptome data was acquired to find differentially expressed genes, and target genes of PfNSUN1 protein were identified by RNA immunoprecipitation and high-throughput sequencing experiments.ResultsThe efficiency of PfNSUN1 protein down-regulated was about 34%. RNA-seq data revealed that differentially expressed genes were mainly down-regulated. And there were 224, 278, 556 genes that were down-regulated with more than 2-fold changes and p-adj<0.05 at ring, trophozoite and schizont stages, respectively. PfNSUN1 protein was significantly enriched on 154 target genes, including 28S ribosomal RNA and pfap2-g5 transcription factor.DiscussionPfNSUN1 is a crucial RNA post-transcriptional modification protein in P. falciparum. It plays a pivotal role in regulating gene expression and parasite growth by targeting 28S ribosomal RNA and pfap2-g5 transcription factor.

5-methylcytosine (m5C) is an important epitranscriptomic modification involved in messenger RNA (mRNA) stability and translation efficiency in various biological processes. However, it remains unclear if m5C modification contributes to the dynamic regulation of the transcriptome during the developmental cycles of Plasmodium parasites. Here, we characterize the landscape of m5C mRNA modifications at single nucleotide resolution in the asexual replication stages and gametocyte sexual stages of rodent (Plasmodium yoelii) and human (Plasmodium falciparum) malaria parasites. While different representations of m5C-modified mRNAs are associated with the different stages, the abundance of the m5C marker is strikingly enhanced in the transcriptomes of gametocytes. Our results show that m5C modifications confer stability to the Plasmodium transcripts and that a Plasmodium ortholog of NSUN2 is a major mRNA m5C methyltransferase in malaria parasites. Upon knockout of P. yoelii nsun2 (pynsun2), marked reductions of m5C modification were observed in a panel of gametocytogenesisassociated transcripts. These reductions correlated with impaired gametocyte production in the knockout rodent malaria parasites. Restoration of the nsun2 gene in the knockout parasites rescued the gametocyte production phenotype as well as m5C modification of the gametocytogenesis-associated transcripts. Together with the mRNA m5C profiles for two species of Plasmodium, our findings demonstrate a major role for NSUN2-mediated m5C modifications in mRNA transcript stability and sexual differentiation in malaria parasites.

The three-dimensional (3D) genome organization plays a critical role in the regulation of gene expression in eukaryotic organisms. In the unicellular malaria parasite Plasmodium falciparum, the high-order chromosome organization has emerged as an important epigenetic pathway mediating gene expression, particularly for virulence genes, but the related architectural factors and underlying mechanism remain elusive. Herein, we have identified the high-mobility-group protein HMGB1 as a critical architectural factor for maintenance of genome organization in P. falciparum. Genome-wide occupancy analysis (chromatin immunoprecipitation sequencing [ChIP-seq]) shows that the HMGB1 protein is recruited mainly to centromeric regions likely via a DNA-binding-independent pathway. Chromosome conformation capture coupled with next-generation sequencing (Hi-C-seq) and 3D modeling analysis show that the loss of HMGB1 disrupts the integrity of centromere/telomere-based chromosome organization accompanied with diminished interaction frequency among centromere clusters. This triggers local chromatin alteration and dysregulated gene expression. Notably, the entire repertoire of the primary virulence genes (var) was completely silenced in the absence of P. falciparum HMGB1 (PfHMGB1). Furthermore, the disrupted nuclear organization was reconstituted by complementation of HMGB1, thereby rescuing the mutually exclusive expression of the var gene family. Collectively, these data demonstrate that the architectural factor HMGB1 is associated with gene expression via mediating the high-order structure of genome organization. This finding not only contributes better understanding of the epigenetic regulation of gene expression but may also provide novel targets for antimalarial strategies. IMPORTANCE Malaria remains a major public health and economic burden currently. The mutually exclusive expression of the virulence genes is associated with the pathogenesis and immune evasion of human malaria parasites in the host. The nuclear architecture provides a well-organized environment for differential gene expression in the nucleus, but the underlying mechanism remains largely unknown. In this study, we have identified the highly conserved high-mobility-group protein HMGB1 as a key architecture regulator involved in virulence gene expression by establishing high-order genome organization in the nucleus of P. falciparum. Mechanistic investigation revealed that the specific interaction of HMGB1 and centromeres constructed the precisely organized nuclear architecture, which coordinated with local chromatin structure to control the singular expression of virulence genes. Hence, this protein appears to be a critical architectural regulator for the pathogenesis of malaria infection and may be a new target for the development of an intervention strategy against malaria.

Identification of RNA targets of RNA-binding proteins (RBPs) is essential for complete understanding of their biological functions. However, it is still a challenge to identify the biologically relevant targets of RBPs through in vitro strategies of RIP-seq, HITS-CLIP, or GoldCLIP due to the potentially high background and complicated manipulation. In malaria parasites, RIP-seq and gene disruption are the few tools available currently for identification of RBP targets. Here, we have adopted the TRIBE (Targets of RNA binding proteins identified by editing) system to in vivo identify the RNA targets of PfDis3, a key exoribonuclease subunit of RNA exosome in Plasmodium falciparum. We generated a transgenic parasite line of PfDis3-ADARcd, which catalyzes an adenosine (A)-to-inosine (I) conversion at the potential interacting sites of PfDis3-targeting RNAs. Most of PfDis3 target genes contain one edit site. The majority of the edit sites detected by PfDis3-TRIBE locate in exons and spread across the entire coding regions. The nucleotides adjacent to the edit sites contain ∼75% of A + T. PfDis3-TRIBE target genes are biases toward higher RIP enrichment, suggesting that PfDis3-TRIBE preferentially detects stronger PfDis3 RIP targets. Collectively, PfDis3-TRIBE is a favorable tool to identify in vivo target genes of RBP with high efficiency and reproducibility. Additionally, the PfDis3-targeting genes are involved in stage-related biological processes during the blood-stage development. Thus PfDis3 appears to shape the dynamic transcriptional transcriptome of malaria parasites through post-transcriptional degradation of a variety of unwanted transcripts from both strands in the asexual blood stage.