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Global transcriptional changes upon Bromodomain (BrD) deletion in PfGCN5 and PHD domain deletion in PfPHD1 by transcriptome analyses via RNA-seq

ABSTRACT: 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.

ORGANISM(S): Plasmodium falciparum

PROVIDER: GSE164070 | GEO | 2020/12/31

REPOSITORIES: GEO

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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:Ubiquitylation is a common post translational modification of eukaryotic proteins. Protein ubiquitylation increases in the transition from intracellular schizont to extracellular merozoite in the asexual blood stage cycle of the human malaria parasite, Plasmodium falciparum (Pf). Here, we identify specific ubiquitylation sites of protein substrates in three intracellular parasite stages and extracellular merozoites; a total of 1464 sites in 546 proteins were identified. 469 ubiquitylated proteins were identified in merozoites compared with only 160 in the preceding intracellular schizont stage, indicating a large increase in protein ubiquitylation associated with merozoite maturation. Following merozoite invasion of erythrocytes, few ubiquitylated proteins were detected in the first intracellular ring stage but as parasites matured through trophozoite to schizont stages the extent of ubiquitylation increased. We identified commonly used ubiquitylation motifs and groups of ubiquitylated proteins in specific areas of cellular function, for example merozoite pellicle proteins involved in erythrocyte invasion, exported proteins, and histones. To investigate the importance of ubiquitylation we screened ubiquitin pathway inhibitors in a parasite growth assay and identified the ubiquitin activating enzyme (UBA1 or E1) inhibitor, MLN7243 (TAK-243) to be particularly effective. This small molecule was shown to be a potent inhibitor of recombinant PfUBA1, and a structural homology model of MLN7243 bound to the parasite enzyme highlights avenues for the development of P. falciparum specific inhibitors. We constructed a genetically modified parasite with a rapamycin-inducible functional deletion of uba1; addition of either MLN7243 or rapamycin to the recombinant parasite line resulted in the same phenotype, with parasite development blocked at the schizont stage. These results indicate that the intracellular target of MLN7243 is UBA1, and this activity is essential for the final differentiation of schizonts to merozoites. The ubiquitylation of many merozoite proteins and their disappearance in ring stages are consistent with the idea that ubiquitylation leads to their destruction via the proteasome once their function is complete following invasion, which would allow amino acid recycling in the period prior to the parasite’s elaboration of a new food vacuole.

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Global transcriptional changes upon Bromodomain (BrD) deletion in PfGCN5 and PHD domain deletion in PfPHD1 by transcriptome analyses via RNA-seq

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