Project description:Three potential ELAV-like proteins of T. brucei, including Tb927.3.2930, Tb927.7.5380, and Tb927.8.6650, were either inhibited by RNAi or phenotypically activated by over-expression, followed by microarray analysis of the transcriptome. The results indicated that these ELAV-like proteins regulate the abundance of a large number of T. brucei transcripts, potentially through regulation of mRNA stability. Each of the three ELAV-like proteins were either inhibited by RNAi or over-expressed, in stable transgenic procyclic form cell lines. Total RNA was extracted 48h after tetracycline induction of the constructs (except for total RNA from Tb927.8.6650 RNAi which was extracted 24h after tet-induction), and sent to NimbleGen for cDNA synthesis and hybridization. Non-induced cells were analyzed in parallel.
Project description:A tagged ectopic version of the ELAV-like protein Tb927.8.6650 of T. brucei was expressed in stable cell lines and pulled down. Co-purifying transcripts were analyzed by sequencing to identify RNAs associated with Tb927.8.6650. Stable procyclic form cell lines expressing tetracycline-inducible TAP-tagged Tb927.8.6650 were created. Cells were harvested 48h after tet-induction, followed by tandem affinity purification of Tb927.8.6650, extraction of co-purified RNA, and sequencing.
Project description:A procyclic form Trypansome brucei RNAi line (PTT parental line, transfected with pALC14 incorporating a TbNMD3 gene fragment) capable of inducing depletion of TbNMD3 was analysed for mRNA expression by RNAseq Cells were grown for 72 hours in culture; RNAi was induced in cells by the addition of 1 microgram/ml of tetracycline
Project description:The first 4 samples belong to the RNA-IP using in situ TAP tagged ZC3H30 in procyclic (insect) form of the parasite T. brucei Lister 427, 2 samples are Elu or eluate, and 2 are FL or flowthrough (unbound) sample. The other 8 samples are also from procyclic cells. 4 samples belong to DKO(ZC3H30 gene double knockout), 2 are non-stressed and 2 are heat shocked samples; the rest 4 samples are DKO-ectopic (ZC3H30 double knockouts, expressing, ectopic copy of ZC3H30) 2 are non-stressed and 2 are heat shocked samples. Heat Shock experiment was done at 39 degree Celsius.
Project description:A procyclic form Trypansome brucei RNAi line (PTT parental line, transfected with pALC14 incorporating a TbNMD3 gene fragment) capable of inducing depletion of TbNMD3 was analysed for mRNA expression by RNAseq
Project description:Epitope tagging and magnetic bead enrichment were combined with SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the parasite Trypanosoma brucei.
Project description:Trypanosoma brucei is a kinetoplastid protozoan and the causal agent of Human African Trypanosomiasis. The life cycle is complex with multiple developmental forms. To identify gene regulations in different developmental forms, we carried out gene expression analysis of two replicative developmental forms which are readily recapitulated in laboratory culture: the ‘slender’ mammalian bloodstream form and the tsetse procyclic form.
Project description:RBP10 was induced for 24 or 48 hours in procyclic cells derived from EATRO1125; this primes the procyclic cells to differentiate to bloodstream form. RNA-seq was performed to quantify mRNAs changes during the differentiation triggered by RBP10 overexpression.
Project description:The pathogenic protozoan T. brucei alternates into distinct developmental stages in the mammalian and insect hosts. The mitogen-activated protein kinase (MAPK) signaling pathways transduce extracellular stimuli into a range of cellular responses, which ultimately lead to the adaptation to the external environment. Here, we combined a loss of function approach with stable isotope labelling with amino acids in cell culture (SILAC)-based mass spectrometry (MS) to investigate the role of the mitogen-activated kinase kinase 5 (MKK5) in T. brucei. The silencing of MKK5 significantly decreased the proliferation of procyclic forms of T. brucei. To shed light into the molecular alterations associated with this phenotype, we measured the total proteome and phosphoproteome of cells silenced for MKK5. In the total proteome, we observed a general decrease in proteins related to ribosome and translation as well as down-regulation of several components of the fatty acid biosynthesis pathway. In addition, we observed alterations in the protein levels and phosphorylation of key metabolic enzymes, which point toward a suppression of the oxidative metabolism. Taken together, our findings show that the silencing of MKK5 alters cell growth, energy metabolism, protein and fatty acid biosynthesis in procyclic T. brucei.