SRA

Purpose: In malaria parasites, the regulation of mRNA translation, storage and degradation is a critical aspect of gene expression, especially during the life stage transition. Since the DEAD-box helicases are involved in RNA metabolism, we wanted to determine whether pfdozi disruption led to altered mRNA abundance in P. falciparum. Methods: In this study, we functionally characterized the DEAD-box RNA helicase PfDOZI in the human malaria parasite Plasmodium falciparum and discovered its functions on mRNA metabolism during development. We generate a pfdozi gene knockout (KO) parasite line. 1). The transcriptomic analysis via RNAseq to the asexual parasites at four stages (ring, early trophozoite, late trophozoite, and schizont)(10h, 20h, 30h, and 40h post-invasion) and the sexual stage (gametocytes) between KO and wildtype (WT) 3D7 parasite line were performed in triplicate. Total RNA was isolated from the purified parasites using the Quick-RNA MiniPrep kit (Zymo Research). RNA sequencing libraries were prepared using the KAPA stranded RNA-seq library preparation kit (Roche) with 500 ng RNA from each sample. Illumina adapter sequence removal and quality trimming of reads were performed using Trimmomatic. Only reads that had a minimum length of 50 base pairs were retained. Reads were then mapped to the P. falciparum 3D7 strain reference genome with HISAT2. Differentially expressed genes are determined by DESeq2 with Padj < 0.01 and absolute log2 fold change higher than 1. 2). To investigate the function of pfdozi in stress response, both KO and WT parasites at the schizont stage were cultured under stress conditions. RNAs were harvested for the transcriptomic analysis via RNAseq. 3). To identify the mRNA targets of PfDOZI in schizonts and gametocytes, we performed RNA-immunoprecipitation (RIP) coupled with next-generation sequencing from the PfDOZI::GFP parasites. A gaussian mixture model with a set of stringent criteria, including a read-depth coverage greater than 10, a fold enrichment ratio greater than 2, and posterior probabilities higher than 0.95 in both replicates. Results: 1). RNA-seq analysis identified up-regulation of 18, 4, 7 and 130 genes and down-regulation of 104, 44, 28 and 15 genes in the ?pfdozi parasite compared to WT 3D7 (fold change = 2 and adjusted P (Padj) < 0.01) at ring (10h), early trophozoite (20h), late trophozoite (30h) and schizont (40h) stage, respectively. Our results showed that pfdozi disruption led to the upregulation of invasion-related genes in schizonts; 2). RNA-seq analysis revealed that the effect of pfdozi disruption on gametocytes was substantially more profound than in asexual stages, with 842 downregulated and 752 upregulated transcripts (fold change =2, Padj < 0.01) in the ?pfdozi gametocyte. About 80% of these markedly reduced transcripts are gametocyte/ookinete-specific transcripts. Genes related to microtubule/cytoskeleton, cellular respiration, and crystalloid were significantly enriched among the downregulated genes, which agrees with the defective morphogenesis of the ?pfdozi gametocytes. 3). RNAseq analysis showed that under the stress conditions, the WT parasites showed three-fold more upregulated transcripts (591) than downregulated (149), suggesting that significantly more transcripts were protected from degradation under stress conditions. In contrast, the ?pfdozi parasites had a much higher proportion (57%, 463) of downregulated than that of upregulated transcripts (43%, 346) under such stress conditions. 4). The RIP-seq analysis collectively identified the potential target mRNAs of the PfDOZI complex(es) in P. falciparum schizonts and gametocytes, identified 823 and 1377 transcripts as potential target mRNAs of the PfDOZI complexes in schizonts and gametocytes, respectively. Our data also showed significant overlaps with genes whose expression was disturbed upon pfdozi disruption. Conclusions: Collectively, we have demonstrated that PfDOZI, likes its metazoan orthologs, is a versatile regulator of mRNA metabolism, and these functions are dynamically regulated during development with stage-specific characteristics. Overall design: Expression profiling of ?pfdozi parasites at different stages or stress conditions, and mRNA targets of pfdozi