Comparative Total RNA-seq between WT and alba4-null Plasmodium yoelii parasites
Ontology highlight
ABSTRACT:
ORGANISM(S): Plasmodium yoelii
PROVIDER: GSE81834 | GEO | 2017/08/25
SECONDARY ACCESSION(S): PRJNA322665
REPOSITORIES: GEO
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Project description:Regulation of Puf2 Expression is Critical for Plasmodium Sporozoite Development and Maintenance of Sporozoite Transmissibility
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Project description:RNA-binding proteins, such as DOZI/CITH and Puf2, have been shown to play critical roles in the life cycle in Plasmodium species. One of the characterized functions of these RNA-binding proteins is to bind to mRNAs and regulate their fates, and it is hypothesized that this regulation of mRNA homeostasis of specific transcripts is important for successful infection of both vectors and hosts by these parasites. To further understand the role that other RNA-binding proteins may play in translational repression and other critical processes in the parasite, we investigated ALBA4, another RNA-binding protein implicated in translational repression. We appended a C-terminal GFP-tag to ALBA4 (ALBA4::GFP), and then performed immunoprecipitations on chemically cross-linked samples using Streptavidin-coated beads coated with a biotin-conjugated anti-GFP antibody to capture the ALBA4 complex(es). We performed these experiments in multiple life cycle stages, including sexual stages (gametocytes) and asexual stage (schizonts). In gametocytes, we determined that ALBA4 associates with translational repression machinery. This is also the case in schizonts, however ALBA4 also associates with complexes involved in active translation, mRNA export, and mRNA degradation. We hypothesize that ALBA4 plays a multi-faceted role in mRNA homeostasis by associating with multiple protein complexes in a stage-specific manner.
2017-08-28 | PXD004183 | Pride
Project description:RNA-binding proteins, such as DOZI/CITH and Puf2, have been shown to play critical roles in the life cycle in Plasmodium species. One of the characterized functions of these RNA-binding proteins is to bind to mRNAs and regulate their fates, and it is hypothesized that this regulation of mRNA homeostasis of specific transcripts is important for successful infection of both vectors and hosts by these parasites. To further understand the role that other RNA-binding proteins may play in translational repression and other critical processes in the parasite, we investigated ALBA4, another RNA-binding protein implicated in translational repression. We appended a C-terminal GFP-tag to ALBA4 (ALBA4::GFP), and then performed immunoprecipitations on chemically cross-linked samples using Streptavidin-coated beads coated with a biotin-conjugated anti-GFP antibody to capture the ALBA4 complex(es). We performed these experiments in multiple life cycle stages, including sexual stages (gametocytes) and asexual stage (schizonts). In gametocytes, we determined that ALBA4 associates with translational repression machinery. This is also the case in schizonts, however ALBA4 also associates with complexes involved in active translation, mRNA export, and mRNA degradation. We hypothesize that ALBA4 plays a multi-faceted role in mRNA homeostasis by associating with multiple protein complexes in a stage-specific manner.
2017-08-28 | PXD006276 | Pride
Project description:High Mobility Group protein HMGB2 is a Critical Regulator of Plasmodium Oocyst Development
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Project description:Sporozoite-specific genes were induced in parasites that express AP2-O whose AP2 domain was swapped with that of AP2-Sp. DNA construct encoding AP2 domain of AP2-Sp was introduced into P. bergei schizonts. It was integrated into the locus of the endogenious AP2-O gene, resulting in swap of the AP2-domain (AP2-O::Sp parasites). Gene expression was analyzed in ookinetes cultured for 20h.
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Project description:Proteomic analysis of Plasmodium yoelii oocyst sporozoites
2019-10-02 | PXD009729 | Pride
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.
2024-04-05 | GSE189034 | GEO
Project description:In malaria parasites, the regulation of mRNA translation, storage and degradation during development and life stage transitions remains largely unknown. Here, we functionally characterized the DEAD-box RNA helicase PfDOZI in P. falciparum. Disruption of pfdozi enhanced asexual proliferation but reduced sexual commitment and impaired gametocyte development. By quantitative transcriptomics, we show that PfDOZI is involved in the regulation of invasion-related genes and sexual stage-specific genes during different developmental stages. PfDOZI predominantly participates in processing body-like mRNPs in schizonts but germ cell granule-like mRNPs in gametocytes to impose opposing actions of degradation and protection on different mRNA targets. We further show the formation of stress granule-like mRNPs during nutritional deprivation, highlighting an essential role of PfDOZI-associated mRNPs in stress response. Here, we demonstrate that PfDOZI participates in distinct mRNPs to maintain mRNA homeostasis in response to life-stage transition and environmental changes by differentially executing post-transcriptional regulation on the target mRNAs.
2024-04-30 | PXD051782 | Pride
Project description:The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the liberation of the parasite from the parasitophorpous vacuole membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, PKA, CK2 and Src-family kinases, are known to be constitutively activated in Theileria infected cells and to contribute to the transformed phenotype. A number of host cell molecules, including the IB kinase and polo-like kinase 1 (Plk1), are recruited to the schizont surface, although very little is known about the schizont molecules involved. In this study we used immunofluorescence analysis to detect phosphorylated threonine (p-Thr and p-Thr-Pro) and serine (p-Ser) epitopes on the schizont during host cell cycle progression, revealing extensive schizont surface phosphorylation during host cell interphase. Further, we isolated schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus mayight be targets forof host cell kinases. In particular, we describe the cell-cycle stage phosphorylation of two T. annulata surface expressed proteins, TaSP and p104. Phospho-peptides of both proteins were significantly more abundant in schizonts purified from S-phase cells.
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