Long non-coding RNAs detected in Plasmodium falciparum malaria using high-resolution DNA tiling microarray technology.
ABSTRACT: Survey of transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. 872 protein coding genes and 60 putative P. falciparum lncRNAs identified under developmental regulation during the parasite's pathogenic human blood stage. Overall design: Total RNA was isolated from synchronous in vitro parasite cultures at 18, 24, 30, and 36 hours post invasion (hpi) of red blood cells. 1 μg of total RNA was then subjected to poly-A selective amplification using Message Amp II (Ambion), substituting biased dNTP/NTP mixes (2A/T/U:1C/G). The resulting aRNA was labeled with Superscript II reverse transcriptase (Invitrogen), using random hexamers, and either Cy3- or Cy5-dUTPs (Amersham).
INSTRUMENT(S): Rinn Sabeti P. falciparum Tiling 385k v1.0
Project description:Survey of transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. 872 protein coding genes and 60 putative P. falciparum lncRNAs identified under developmental regulation during the parasite's pathogenic human blood stage. Total RNA was isolated from synchronous in vitro parasite cultures at 18, 24, 30, and 36 hours post invasion (hpi) of red blood cells. 1 μg of total RNA was then subjected to poly-A selective amplification using Message Amp II (Ambion), substituting biased dNTP/NTP mixes (2A/T/U:1C/G). The resulting aRNA was labeled with Superscript II reverse transcriptase (Invitrogen), using random hexamers, and either Cy3- or Cy5-dUTPs (Amersham).
Project description:Epigenetic mechanisms have been poorly understood in Plasmodium falciparum, the causative agent of malaria. To elucidate stage specific epigenetic regulations in P. falciparum, we performed genome-wide mapping of various histone modifications, nucleosomes and RNA Polymerase II. Our comprehensive analysis suggest that transcription initiation and elongation are distinct in Plasmodium. In this study, by analyzing histone modifications, nucleosome occupancy and RNA Polymerase II (Pol II) at three different IEC developmental stages of Plasmodium; ring, trophozoite and schizont, we tried to unravel the epigenetic mechanism associated with gene regulation. Examination of H3K27me3, H3K4me3, H3K9me3, H3K14ac, H3K4me1, H3K79me3, H3K27ac, H3K4me2, H3K9ac, H4ac, RNA Pol II and Histone H3 at three different stages of Plasmodium falciparum
Project description:Gene expression in Plasmodium falciparum is tightly regulated to ensure successful propagation of the parasite throughout its complex life cycle. The earliest transcriptomics studies in P. falciparum suggested a cascade of transcriptional activity over the course of the 48-hour intraerythrocytic developmental cycle (IDC); however, the just-in-time transcriptional model has recently been challenged by findings that show the importance of post-transcriptional regulation. To further explore the role of transcriptional regulation, we performed the first genome-wide nascent RNA profiling in P. falciparum. Our findings indicate that the majority of genes are transcribed simultaneously during the trophozoite stage of the IDC and that only a small subset of genes is subject to differential transcriptional timing. RNA polymerase II is engaged with promoter regions prior to this transcriptional burst, suggesting that Pol II pausing plays a dominant role in gene regulation. In addition, we found that the overall transcriptional program during gametocyte differentiation is surprisingly similar to the IDC, with the exception of relatively small subsets of genes. Results from this study suggest that further characterization of the molecular players that regulate stage-specific gene expression and Pol II pausing will contribute to our continuous search for novel antimalarial drug targets. Overall design: Analysis of transcriptional activity of P. falciparum during erythrocytic stages using newly synthesized RNA, nascent RNA. In addition, RNA polymerase II occupancy was explored and used to verify findings discovered using nascent RNA data set.
Project description:Epstein-Barr virus (EBV) causes Burkitt, Hodgkin, and post-transplant B-cell lymphomas. How EBV remodels metabolic pathways to support rapid B-cell outgrowth remains largely unknown. To gain insights, primary human B-cells were profiled by tandem-mass-tag based proteomics at rest and at 9 time points after infection. >8000 host and 29 viral proteins were quantified, revealing mitochondrial remodeling and induction of one-carbon (1C) metabolism. EBV-encoded EBNA2 and its target MYC were required for upregulation of the central mitochondrial 1C enzyme MTHFD2, which played key roles in EBV-driven B-cell growth and survival. MTHFD2 was critical for maintaining elevated NADPH levels in infected cells, and oxidation of mitochondrial NADPH diminished B-cell proliferation. Tracing studies underscored contributions of 1C to nucleotide synthesis, NADPH production and redox defense. EBV upregulated import and synthesis of serine to augment 1C flux. Our results highlight EBV-induced 1C as a potential therapeutic target and provide a new paradigm for viral onco-metabolism.
Project description:Nuclear-localized RNA binding proteins are involved in various aspects of RNA metabolism, which in turn modulates gene expression. However, the functions of nuclear-localized RNA binding proteins in plants are poorly understood. Here we report the functions of two proteins containing RNA recognition motifs, At RZ-1B and At RZ-1C, in Arabidopsis. At RZ-1B and At RZ-1C were localized to nuclear speckles and interacted with a spectrum of serine/arginine-rich (SR) proteins through their C-termini. At RZ-1C preferentially bound to purine-rich RNA sequences in vitro through its N-terminal RNA recognition motif. Disrupting the RNA-binding activity of At RZ-1C with SR proteins through over-expression of the C-terminus of At RZ-1C conferred defective phenotypes similar to those observed in At rz-1b/At rz-1c double mutants, including delayed seed germination, reduced stature, and serrated leaves. Loss of function of At RZ-1B and At RZ-1C was accompanied by defective splicing of many genes and global perturbation of gene expression. In addition, we found that At RZ-1C directly targeted FLC, promoting efficient splicing of FLC introns and likely also repressing FLC transcription. Our findings highlight the critical role of At RZ-1B/1C in regulating RNA splicing, gene expression, and many key aspects of plant development via interaction with proteins including SR proteins. mRNA-seq to look at the transcriptome and splicing differences between wild type and At rz-1b At rz-1c mutant of Arabidopsis thaliana
Project description:The natural product taxol is an efficient and worldwide accepted anticancer drug. Taxol and other taxoids accumulate in only a few specific genera of the Taxaceae family, such as Taxus and Pseudotaxus. The comprehensive variations among three Taxaceae trees, including one taxol-producing (TP) and two non-taxol producing (NTP) trees, were compared. In total, 3,655 metabolites and 88,609 functional genes were identified through metabolomic and transcriptomic analyses, respectively. KEGG enrichment analysis showed that many differentially expressed genes were enriched in several hormone-, flavonoid-, and terpenoid-related metabolic pathways. The hormones, abscisate and abscisic acid, highly accumulated in the NTP trees, whereas gibberellic acids substantially accumulated in the TP trees. Overall design: The comprehensive variations among three Taxaceae trees, including one taxol-producing (TP) and two non-taxol producing (NTP) trees, were compared.
Project description:Heterochromatin is a tightly packaged form of DNA that leads to permanent or temporal gene silencing. In P. falciparum heterochromatin is formed after trimethylation of lysine 9 on histone H3 and consequent binding of heterochromatin protein 1 (HP1). Genome-wide profiling studies established that in P. falciparum blood stage schizonts heterochromatin is formed at subtelomeres and some intra-chromosomal islands. Reversible silencing of genes located in these regions has been shown to be key to, among others, antigenic variation, invasion pathway selection or commitment to gametocytogenesis. However, heterochromatic gene silencing has not been investigated in many other Plasmodium species, strains or life cycle stages, yet. Here, we used ChIP-seq to profile HP1 occupancy in asexual parasites of six different species (P. falciparum, P. knowlesi, P. vivax, P. berghei, P. yoelii, and P. chabaudi) and of four different P. falciparum strains (3D7, PF2004, NF54, NF135) as well as two different P. knowlesi clones (A1C1, A1H1). Additionally, we performed HP1 ChIP-seq on three asexual stages (ring, trophozoite and schizont stage) and two sexual stages of P. falciparum parasites. Collectively, the generated HP1 profiles provide a detailed catalog of heterochromatic genes and reveal conserved and specialized features of epigenetic control at different life cycle stages, strains and species across the genus Plasmodium. Overall design: In total, we sequenced 26 samples (14 HP1-ChIP-seq samples and 12 inputs) generated from chromatin from six different Plasmodium species (P. chabaudi, P. berghei, P. yoelii, P. knowlesi, P. vivax and P. falciparum), four different strains of P. falciparum (3D7, NF54, NF135 and Pf2004) and two different strains of P. knowlesi (A1.C1 and A1.H1) as well as three different intraerythrocytic stages of the P. falciparum 3D7 strain (ring, trophozoite and schizont stages) and three different stages of the P. falciparum Pf2004 strain (schizont stages, stage II/III gametocytes and stage IV/V gametocytes).
Project description:Nuclear myosin 1c (NM1) is emerging as regulator of transcription and chromatin organization. Using a genome-wide approach we report here that NM1 binds across the mammalian genome with occupancy peaks at class II gene promoters, correlating with distributions of RNA Polymerase II (Pol II) and active epigenetic marks. We show that NM1 synergizes with polymerase-associated actin to maintain active Pol II at gene promoters. NM1 also co-localizes with the nucleosome remodeler SNF2h at class II promoters where they assemble together with WSTF as part of the B-WICH complex to remodel chromatin. Following B-WICH assembly, NM1 mediates physical recruitment of the histone acetyl transferase PCAF and the histone methyl transferase Set1/Ash2 to maintain and preserve H3K9acetylation and H3K4trimethylation for active transcription. We propose a novel genome-wide mechanism where myosin synergizes with Pol II-associated actin to link the polymerase machinery with permissive chromatin for transcription activation. Association of nuclear myosin 1 (NM1) with the genome in mouse embryonic fibroblasts
Project description:SREBF-1c is a transcription factor regulating fatty acid biosynthesis. We have charaterized the impact of the abcence of SREBF-1c on the development of peripheral neuropathy In this dataset we included expression data from dissected sciatic nerve from 10 months old SREBF-1c KO mice and relative littermates. Overall design: 6 total samples were analyzed (3 wild type and 3 SREBF-1c KO). Data handling was mainly done using TAC and Partek Genomic Suite software. The Robust Multichip Average (RMA) method was employed to calculate probe set intensity. RMA is a robust multi-chip average expression method that estimates the probe-level data from a set of chips: the perfect-match (PM) values are background-corrected, normalized and finally summarized resulting in a set of expression measures. The identification of differentially expressed genes was addressed using Fold Change (FC) and ANOVA. For each experimental condition the probesets whose fold change are higher or equal than 1.5 with an ANOVA unadjusted p-value lower than 0,05 are selected as significantly modulated.
Project description:Eukaryotic gene expression requires that RNA Polymerase II (RNAP II) gain access to DNA in the context of chromatin. The C-terminal domain (CTD) of RNAP II recruits chromatin modifying enzymes to promoters, allowing for transcription initiation or repression. Specific CTD phosphorylation marks facilitate recruitment of chromatin modifiers, transcriptional regulators, and RNA processing factors during the transcription cycle. However, the readable code for recruiting such factors is still not fully defined and how CTD modifications affect related families of genes or regional gene expression is not well understood. Here we examine the effects of manipulating the Y1S2P3T4S5P6S7 heptapeptide repeat of the CTD of RNAP II in Schizosaccharomyces pombe by substituting non-phosphorylatable alanines for Ser2 and/or Ser7 and the phosphomimetic glutamic acid for Ser7. Global gene expression analyses were conducted using splicing-sensitive microarrays and validated via RT-qPCR. The CTD mutations did not affect pre-mRNA splicing or snRNA levels. Rather, the data revealed upregulation of subtelomeric genes and alteration of the repressive histone H3 lysine 9 methylation (HeK9me) landscape. The data further indicate that H3K9me and expression status are not fully correlated, suggestive of CTD-dependent subtelomeric repression mechansims that act independently of H3K9me levels. Splicing sensitive S. pombe microarrays (Agilent-027365) were used to compare the splicing and expression profile of four mutant strains relative to WT control with 3-5 biological replicates and dye flipped samples