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Transcriptiome of ΔPfDNMT parasite during malaria parasite asexual development [DNMT_RNAseq]

ABSTRACT: Purpose: this study is to analyze the change of overal transcriptome after disruption of DNA methyltransferase (DNMT) in Plasmodium falciparum. Methods: In this study, the transcriptomes of a PfDNMT gene knockout (KO) parasite line with its wildtype control, its complementation (adding back the DNMT expression by episomal expression of DNMT in the DNMT KO parasite), and overexpression (episomal expression of DNMT in the wildtype parasite) were analyzed by RNAseq. Total RNA were harvested from the asexual parasites at three developmental stages (ring, trophozoite, and schizont) 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. Results: Using an optimized data analysis workflow, we mapped about 5 million sequence reads per sample to the malaria parasite genome (pf3D7_V3.0.) and identified 5712 transcripts with high mapping rate at the range between 80% and 95. PfDNMT KO profoundly disturbed the global transcription pattern,especially at trophozoite stage, causing 1732 (30.3%) genes to be differentially expressed at trophozoite.Complementation restored the expression pattern and overexpression of PfDNMT caused nearly 2000 gene ro be differentially expressed at schizont stage. Conclusions: Collectively, transcriptomic analysis of DNMT KO, complemetation and overexpression shows PfDNMT plays important role in gene regulation.

ORGANISM(S): Plasmodium falciparum

PROVIDER: GSE199368 | GEO | 2023/05/17

REPOSITORIES: GEO

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Project description:Purpose: In malaria parasite, PfGCN5, a histone acetyltansferase (HAT),forms a unique complex including a PHD domain containing protein named PfPHD1. To understand the function of this complex, the BrD in PfGCN5 and PHD in PfPHD1 were deleted. The transcritional changes after domain deletions were measured by RNA-seq. Methods: The mRNA profiles of parasite lines : 3D7 (WT), PfGCN5-ΔBrD and PfPHD1-ΔPHD, were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500 in Rapid Run mode using 100nt single read sequencing. Three replicates of total RNA from parasites at ring, early trophozoite, late trophozoite and schizont stages were harvested by using the ZYMO RNA purification kit, and used to generate the sequencing libraries using the KAPA Stranded mRNA Seq kit for the Illumina sequencing platform according to the manufacturer's protocol (KAPA biosystems). Quality trimming and adapter sequence was performed using Trimmomatic (v0.36). Low quality bases were trimmed using a sliding window approach with a minimum average quality of 18 in 4 base pair window. The Reads with length <35 base pairs were removed. The trimmed reads were mapped to the P. falciparum genome sequence (Genedb v3.1) using HISAT2 (Kim et al., 2015). The coverage was analyzed by using the bedtools (Quinlan, 2014). The expression levels and the differential expression were calculated by FeatureCounts and DESeq2 (Anders and Huber, 2010; Liao et al., 2014). Results: Using an optimized data analysis workflow, we mapped about 5 million sequence reads per sample to the malaria parasite genome (pf3D7_V3.0.) and identified 5643 transcripts with high mapping rate at the range between 80% and 95. PfGCN5 BrD deletion profoundly disturbed the global transcription pattern, causing 3533 (62.6%) genes to be differentially expressed in at least one of the four time points analyzed. Specifically, BrD deletion resulted in down-regulation of 997, 799, 861 and 902 genes, and up-regulation of 1127, 780, 846, and 368 genes at the ring, early trophozoite, late trophozoite, and schizont stage, respectively. PHD deletion in PfPHD1 caused a similar but more profound disturbance of gene expression during the IDC, with 3870 (68.6%) transcripts being differentially expressed in at least one of the four stages analyzed. The PfPHD1 PHD deletion resulted in the down-regulation of 872, 1021, 557, and 787 genes, and up-regulation of 1481, 1266, 648, and 1028 genes at the ring, early trophozoite, late trophozoite and schizont stage, respectively. Conclusions: Either domain deletion profoundly disturbed the global transcription pattern, causing altered expression in more than 60% of the genes including general cellular pathways such as DNA replication and translation, and parasite-specific pathways such as invasion, cytoadherence, and sexual development.

Project description: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.

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.

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Transcriptiome of ΔPfDNMT parasite during malaria parasite asexual development [DNMT_RNAseq]

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