Identification of the mRNAs associated with the protein complex PuREBP1/PuREBP2 (Tb927.4.4550/Tb927.3.3060) in Trypanosoma brucei [RIP-seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Trypanosoma brucei brucei
PROVIDER: GSE145466 | GEO | 2021/02/01
REPOSITORIES: GEO
Similar Datasets
Project description:We have previously characterized a short RNA stem-loop cis-element within the 3'-UTR of the NT8 nucleobase transporter mRNA (PuRE, Purine Responsive Element) that is necessary and sufficient to confer a strong repression of gene expression in response to purines in the human parasite Trypanosoma brucei (PMID: 24813448). In this study, we have identified a protein complex composed of two RNA-binding proteins (Tb927.4.4550 and Tb927.3.3060, named here as PuREBP1 and PuREBP2 respectively) that binds to the PuRE in vitro and to NT8 mRNA in vivo. Depletion of PuREBP1 by RNAi results in the upregulation of just NT8 and the mRNAs encoding the amino acid transporter AATP6 paralogues. Moreover, we found that the PuREBP1/2 complex is associated with only a handful of mRNAs, and that it is responsible for the observed purine-dependent regulation of NT8 expression. Depletion of PuRBP1 results in the upregulation of just two mRNAs (encoding membrane transporters NT8 and AATP6) and downregulation of four other mRNAs (including the one encoding PuREBP1, as expected)
2021-02-01 | GSE145468 | GEO
Project description:The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and post-translational mechanisms. One mRNA sample from each of Purine-Starved and Purine-Replete cells were analyzed using SL RNA-Seq methodology
2014-02-01 | E-GEOD-48394 | biostudies-arrayexpress
Project description:Purines serve as the building blocks for DNA and RNA, confer cellular energy and signaling. Purines are generated by de novo synthesis pathway and salvage pathway. Under purine-depleted or other cellular stresses, enzymes in the purine de novo synthesis pathway form a dynamic and reversible condensate called purinosome, but the underlying mechanism of purinosome formation is unknown. In this thesis, we found that ASB11-based Cul5 E3 ligase promotes ubiquitination of PAICS, a purine de novo synthesis enzyme. This ubiquitination does not lead to PAICS degradation, but drives purinosome assembly. We provide evidence that purinosome assembly involves a liquid-liquid phase separation (LLPS) process and identify several ubiquitin binding proteins that may bind ubiquitinated PAICS through a multivalent mode to drive LLPS and purinosome assembly. Importantly, ASB11 is upregulated under the stresses that promote purinosome assembly, thus increasing the formation of ASB11/PAICS complex. Finally, we demonstrated that melanoma cells express a high level of ASB11 to confer a constitutive purinosome formation, which support their viability. In summary, our study identifies ASB11-mediated PAICS ubiquitination as a driving mechanism for purinosome assembly, the regulation of this mechanism under stressed conditions, and the importance of this regulation in cell viability.
2023-10-06 | PXD027946 | Pride
Project description:Purines serve as the building blocks for DNA and RNA, confer cellular energy and signaling. Purines are generated by de novo synthesis pathway and salvage pathway. Under purine-depleted or other cellular stresses, enzymes in the purine de novo synthesis pathway form a dynamic and reversible condensate called purinosome, but the underlying mechanism of purinosome formation is unknown. In this thesis, we found that ASB11-based Cul5 E3 ligase promotes ubiquitination of PAICS, a purine de novo synthesis enzyme. This ubiquitination does not lead to PAICS degradation, but drives purinosome assembly. We provide evidence that purinosome assembly involves a liquid-liquid phase separation (LLPS) process and identify several ubiquitin binding proteins that may bind ubiquitinated PAICS through a multivalent mode to drive LLPS and purinosome assembly. Importantly, ASB11 is upregulated under the stresses that promote purinosome assembly, thus increasing the formation of ASB11/PAICS complex. Finally, we demonstrated that melanoma cells express a high level of ASB11 to confer a constitutive purinosome formation, which support their viability. In summary, our study identifies ASB11-mediated PAICS ubiquitination as a driving mechanism for purinosome assembly, the regulation of this mechanism under stressed conditions, and the importance of this regulation in cell viability.
2023-10-06 | PXD038411 | Pride
Project description:The ability of Leishmania to survive in their insect or mammalian host is dependent upon an ability to sense and adapt to changes in the microenvironment. However, little is known about the molecular mechanisms underlying the parasite response to environmental changes, such as nutrient availability. To elucidate nutrient stress response pathways in Leishmania donovani, we have used purine starvation as the paradigm. The salvage of purines from the host milieu is obligatory for parasite replication; nevertheless, purine-starved parasites can persist in culture without supplementary purine for over 3 months, indicating that the response to purine starvation is robust and engenders parasite survival under conditions of extreme scarcity. To understand metabolic reprogramming during purine starvation we have employed global approaches. Whole proteome comparisons between purine-starved and purine-replete parasites over a 6-48 h span have revealed a temporal and coordinated response to purine starvation. Purine transporters and enzymes involved in acquisition at the cell surface are upregulated within a few hours of purine removal from the media, while other key purine salvage components are upregulated later in the time-course and more modestly. After 48 h, the proteome of purine-starved parasites is extensively remodeled and adaptations to purine stress appear tailored to deal with both purine deprivation and general stress. To probe the molecular mechanisms affecting proteome remodeling in response to purine starvation, comparative RNA-seq analyses, qRT-PCR, and luciferase reporter assays were performed on purine-starved versus purine-replete parasites. While the regulation of a minority of proteins tracked with changes at the mRNA level, for many regulated proteins it appears that proteome remodeling during purine stress occurs primarily via translational and post-translational mechanisms.
2014-02-01 | GSE48394 | GEO
Project description:A novel class of translation inhibitors isolated from Aglaia plants, exemplified by Rocaglamide A (RocA), shows promise as an anti-cancer therapy. RocA converts its molecular target, translation initiation factor 4A (eIF4A), a DEAD-box protein, into a purine-selective RNA-binding protein that represses protein synthesis from a subset of mRNAs. This unusual mechanism of action raises the question of how the drug induces selectivity for polypurine sequences. Furthermore, as eIF4A is found in all eukaryotes, it is unclear how Aglaia resists the effects of RocA. Here, we assembled the Aglaia transcriptome de novo and identified highly specific mutations in eIF4A that enable it to evade RocA-dependent translation inhibition. Furthermore, we determined the crystal structure of the human eIF4A1•ATP analog•RocA•polypurine RNA complex. RocA binds residues mutated in Aglaia and intercalates between consecutive purines at a sharp RNA bend, revealing the structural basis of polypurine-selective translational inhibition by RocA in human and resistance in Aglaia.
2018-12-30 | GSE102720 | GEO
Project description:Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase (PFAS), a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking PFAS or phosphoribosyl pyrophosphate amidotransferase (PPAT), another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mTORC1 suppression, and was profoundly suppressed in cells deficient for TSC2, which negatively regulates mTORC1 through inhibition of RHEB, suggesting that purines regulate autophagy through the TSC-RHEB-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) and dihydroorotate dehydrogenase (DHODH) activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.
2021-05-11 | GSE164778 | GEO
Project description:The purine signaling pathway is crucial for cellular function and is a conserved metabolic network from prokaryotes to humans. While extensively studied in microorganisms like yeast and bacteria, the impact of perturbing dietary intermediates from the purine signaling pathway on animal development and growth remains poorly understood. We utilized Caenorhabditis elegans as the metazoan model to investigate the mechanisms underlying this deficiency. Through a high-throughput screening of an E. coli mutant library Keio collection, we identified 34 E. coli mutants that delay C. elegans development. Among these mutants, we found that E. coli purE gene is an essential genetic component that promotes host development in a dose-dependent manner. Further metabolites supplementation suggests that bacterial purE downstream metabolite 5-aminoimidazole-4-carboxamide ribotide (AICAR) can inhibit worm growth. Additionally, we found the FoxO transcription factor DAF-16 is indispensable in worm development delay induced by purE mutation, and observed increased nuclear accumulation of DAF-16 when fed E. coli purE- mutants, suggesting the role of DAF-16 in response to purE mutation. RNA-seq analysis and phenotypic assays revealed that worms fed the E. coli purE mutant exhibited elevated lifespan, thermotolerance, and pathogen resistance. These findings collectively suggest that certain intermediates in the bacterial purine signaling pathway can serve as a cue to modulate development and activate the defense response in the nematode C. elegans through DAF-16.
2024-10-06 | GSE278640 | GEO
Project description:Shortening of polyadenosine (poly(A)) tails (deadenylation) is the initial step in the decay of most mRNAs. The CCR4-NOT complex is the major deadenylase in mammals. Interaction of Cnot1 with RNA binding proteins, including the miRNA-induced silencing complex (miRISC), which includes Argonaute (Ago) and GW182 as core proteins, and the TTP family of AU-rich element (ARE)-binding proteins (TTP and BRF1/2), recruits the CCR4-NOT complex to the 3’-untranslated regions (3’-UTR) of mRNAs, leading to their degradation. This experiment aimed to determine the binding affinity of the CCR4-NOT complex, Ago2, and BRF1 with their target mRNAs in liver. Liver isolated from C57BL/6J wild-type mice at 8 weeks of age was solubilized in TNE buffer for 30 min at 4ºC. Lysate was incubated with antibodies against Cnot3, Ago2, and BRF1 for 1 hr at 4ºC, and then incubated with Dynabeads (Invitrogen) for 2 hr at 4ºC. mRNAs in immune complexes were isolated using Isogen II. 100 ng of total RNA was used for RNA-seq library preparation with TruSeq Stranded mRNA Library Prep Kit for NeoPrep. 150 base-pair pair-end read RNA-seq was performed with Hiseq 3000/4000 PE Cluster Kit and Hiseq 3000/4000 SBS Kit on Hiseq4000.
2020-03-30 | E-MTAB-6941 | biostudies-arrayexpress
Project description:Transcriptional profiles of Escherichia coli MG1655 in mixed culture with Pseudomonas aeruginosa PAO1 showed a number of E. coli genes to be upregulated including purA-F and other genes associated with purine synthesis. In contrast, genes associated with pyrimidine synthesis were unaffected. Competition experiments in both planktonic and biofilm cultures, using three purine synthesis mutants, purD, purH, and purT showed little difference in E. coli survival from the parent strain. As purines are components of the cell signals, cAMP and c-di-GMP, we conducted competition experiments with E. coli mutants lacking adenylate cyclase (cyaA), cAMP phosphodiesterase (cpdA), and the catabolite receptor protein (crp), as well as ydeH, an uncharacterized gene that has been associated with c-di-GMP synthesis. Survival of the cyaA and crp mutants during co-culture were significantly less than the parent strain. Supplementation of the media with 1mM cAMP could restore survival of the cyaA mutant but not the crp mutant. In contrast, survival of the cpdA mutant was similar to the parent strain. Survival of the ydeH mutant was moderately less than the parent, suggesting that cAMP has more impact on E. coli mixed culture growth than c-di-GMP. Addition of 1 mM indole restored the survival of both the cyaA and crp mutations. Mutants in genes for tryptophan synthesis (trpE) and indole production (tnaA) showed a loss of competition and recovery through indole supplementation, comparable to the cyaA and crp mutants. Overall, these results suggest indole and cAMP as major contributing factors to E. coli growth in mixed culture. Two replicates of LB grown E. coli ZK126 and P. aeruginosa PAO1 were grown in pure culture at 37C to an OD of 0.3 (log phase) and then mixed 1:1 and incubated for an additional 45 min. RNA was extracted, purified, reverse transcribed to cDNA and then analyzed on E. coli and P. aeruginosa chips from Affymetrix. Expression profiles were compared to pure culture E. coli and P. aeruginosa grown on LB.
2011-11-09 | E-GEOD-26931 | biostudies-arrayexpress