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Global transcriptional changes of Δpfdozi parasite in different stages and conditions

ABSTRACT: 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 Δpfdozi 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 Δpfdozi 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 Δpfdozi 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 Δpfdozi 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.

ORGANISM(S): Plasmodium falciparum

PROVIDER: GSE189034 | GEO | 2024/04/05

REPOSITORIES: GEO

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Project description:The human malaria parasite Plasmodium falciparum has a complex and multi-stage life cycle that requires extensive immune escape, invasion of human liver and blood cells, and transmission through the female Anopholes mosquito. To date, the regulatory elements orchestrating these critical parasite processes remain largely unknown. However, there is mounting evidence across a broad range of species that intergenic long non-coding RNA (lncRNA) and antisense RNA can regulate chromatin state and gene expression. To pursue such functional roles for lncRNAs in P. falciparum, we performed deep, strand-specific RNA sequencing of fifteen non-polyA-selected blood stage samples, and assembled and characterized the properties of 660 intergenic lncRNAs, 474 antisense RNAs, and 1381 circular RNAs (circRNAs). We further validated the non-canonical splice junctions of seven P. falciparum circRNAs, an emerging class of non-coding RNA with regulatory potential and unexplored functional significance in P. falciparum. Our comprehensive analysis of P. falciparum lncRNAs indicates a functional role for these transcripts; P. falciparum intergenic lncRNAs and antisense RNAs are developmentally regulated in a similar periodic fashion to annotated transcripts, and sense-antisense pair expression is significantly anti-correlated. Notable outliers include intergenic lncRNAs that strongly peak in expression during parasite invasion, such as the telomere-associated lncRNA-TARE family, antisense transcripts that drop in expression during parasite invasion, and a highly correlated, multi-exonic, antisense counterpart to P. falciparum Gametocyte Developmental Protein 1 (PfGDV1). Taken together, our results present over two thousand P. falciparum intergenic lncRNA, antisense, and circRNA candidates and highlight promising P. falciparum lncRNAs for future investigation. We harvested fifteen blood stage samples from two biological replicate time-courses. The first time-course comprised of eleven samples that finely map temporal changes during P. falciparum blood stage development. We harvested samples over 56 hours, at roughly 4-hour time intervals, from a tightly synchronized P. falciparum 3D7 parasite population. As the asexual blood stage is an approximately 48-hour cycle, this time-course allowed us to profile gene expression during RBC rupture and parasite invasion. The second time-course comprised of four samples harvested in synchronous P. falciparum 3D7 parasites approximately four hours before and after the ring to trophozoite and trophozoite to schizont morphological stage transitions, which occur during the blood stage at 24 hours post invasion (hpi) and 36 hpi, respectively.

Project description:For malaria transmission, the parasite must undergo sexual differentiation into mature gametocytes. However, the molecular basis for this critical transition in the parasites life cycle is unknown. Six previously uncharacterized genes, Pfg14.744, Pfg14.745, Pfg14.748, Pfg14.763, Pfg14.752 and Pfg6.6 that are members of a 36 gene Plasmodium falciparum-specific subtelomeric superfamily were found to be expressed in parasites that are committed to sexual development as suggested by co-expression of Pfs16 and Pfg27. Northern blots demonstrated that Pfg14.744 and Pfg14.748 were first expressed before the parasites differentiated into morphologically distinct gametocytes, transcription continued to increase until stage II gametocytes were formed and then rapidly decreased. Immunofluorescence assays indicated that both proteins were only produced in the subpopulation of ring stage parasites that are committed to gametocytogenesis and both localized to the parasitophorous vacuole (PV)b of the early ring stage parasites. As the parasites continued to develop Pfg14.748 remained within the parasitophorous vacuole, while Pfg14.744 was detected in the erythrocyte. The 5' flanking region of either gene alone was sufficient to drive early gametocyte specific expression of green fluorescent protein (GFP). In parasites transfected with a plasmid containing the Pfg14.748 5' flanking region immediately upstream of GFP, fluorescence was observed in a small number of schizonts the cycle before stage I gametocytes were observed. This expression pattern is consistent with commitment to sexual differentiation prior to merozoite release and erythrocyte invasion. Further investigation into the role of these genes in the transition from asexual to sexual differentiation could provide new strategies to block malaria transmission. Microarray analysis was used to compare two clones derived from Plasmodium falciparum strain 3D7 parasites that differ in their ability to undergo gametocytogenesis. Clone G+ produces gametocytes and clone G- produces very few if any gametocytes. RNA was harvested from the cultures when the asexual parasitemia was 0.9-1.48% (day 4) (n=4) after setting up the gametocyte cultures and 5.2-5.58% (day 6) (n=4) prior to the appearance of morphologically distinct gametocytes and used to generate cDNA that was labeled with Cy3 or Cy5 and hybridized to the Plasmodium falciparum 70 mer oligonucleotide microarray developed by DeRisi and co-workers.

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Global transcriptional changes of Δpfdozi parasite in different stages and conditions

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