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Caenorhabditis elegans mRBPome

ABSTRACT: This project aims to identify novel RNA binding proteins in the nematode, Caenorhabditis elegans. Since interactions between RNAs and proteins may be transient, these animals were crosslinked with UV light at 254 nm which promotes the covalent link between proteins and RNAs. After this, polyadenylated mRNAs were purified via oligo(dT) coupled to magentic beads under stringent conditions. Finally, samples were subjected to mass spectrometry analysis. To rule out the possibility of RNA-independent binding we also analysed other samples: i) samples digested with RNase one; ii) samples where we performed competition assays with polyadenylic acid

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Caenorhabditis Elegans

SUBMITTER: Ana Matia Gonzalez

LAB HEAD: André P. Gerber

PROVIDER: PXD002226 | Pride | 2015-10-26

REPOSITORIES: Pride

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Publications

Conserved mRNA-binding proteomes in eukaryotic organisms.

Matia-González Ana M AM Laing Emma E EE Gerber André P AP

Nature structural & molecular biology 20151123 12

RNA-binding proteins (RBPs) are essential for post-transcriptional regulation of gene expression. Recent high-throughput screens have dramatically increased the number of experimentally identified RBPs; however, comprehensive identification of RBPs within living organisms is elusive. Here we describe the repertoire of 765 and 594 proteins that reproducibly interact with polyadenylated mRNAs in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively. Furthermore, we report the different ...[more]

PMID: 26595419

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Quantitative temporal phosphoproteomics of aging C.elegans under different environmental temperatures

Project description:Temperature is one of the primary environmental factors that affect aging, in which protein phosphorylation is an important regulator. Currently, the understanding of phosphorylation events in regulatory networks during aging has remained rather limited. Therefore, the phosphoproteomes of C.elegans of different age groups cultured at 20°C in natural aging process and 25°C in accelerated aging process were analyzed. Through using the iTRAQ-labeled phosphoproteomics method, 2375 phosphoproteins and 9063 phosphosites were identified. Volcano plots illustrated that 208 proteins during natural aging and 130 during accelerated aging, were significantly changed. Gene ontology and pathway analysis revealed that these proteins were mainly involved in temperature response, DNA transcription, protein translation, tissue system development and animal behavior processes. Moreover, our results uncovered those kinases CK2, MAPK and CAMK2 might play important roles in aging regulation. In summary, our results provided a new insight into the complicated phosphor-regulatory network system during aging and an important resource for future studies of protein phosphorylation in worms.

2018-08-08 | PXD009661 | Pride

One-day thermal regime extends lifespan

Project description:Environmental factors such as temperature can modulate animal’s lifespan. However, the underlying mechanisms remain largely undefined. Through proteomic analysis of C. elegans at different time points (young adult, middle age, and old age) and under two temperature conditions (20°C and 25°C), we found that temperature affects C. elegans lifespan largely by modulating the proteostasis. Surprisingly, although animals live shorter at the higher temperature, we found that worms cultured at 25°C for one day at early adulthood promotes the protein homeostasis by decreasing the synthesis and increasing the degradation, suggesting the beneficial effects. Consistent with the finding, animals shifting from 20°C to 25°C for 24 hours between the larval L4 to adult Day 3 stages indeed live longer and are more resistant to stresses. Furthermore, we found that the one-day thermal induced lifespan extension is mediated by the FOXO transcriptional factor DAF-16. These results reveal an unexpected and complicated mechanism underlying the temperature effects on aging, which could potentially aid to develop strategies for treating aging related diseases.

2023-03-11 | PXD024916 | Pride

H3K4me2 regulates the recovery of protein biosynthesis and homeostasis from UV-induced DNA damage during development and aging

Project description:DNA damage causes cancer, impairs development and accelerates aging. UV irradiation induces transcription-blocking lesions and defects in transcription-coupled nucleotide excision repair lead to developmental failure and premature aging in humans. Following DNA repair, the homeostatic processes need to be reestablished to ensure development and maintain tissue functionality. Here, we report that in C. elegans removal of the MLL/COMPASS H3K4 methyltransferase exacerbates the developmental growth retardation and accelerates aging, while depletion of the H3K4 demethylase, SPR-5, promotes developmental growth and extends lifespan amid UV-induced damage. We demonstrate that specifically the DDR-induced H3K4me2 is associated with the activation of genes regulating RNA transport, splicing, ribosome biogenesis, and protein homeostasis and regulates the recovery of protein biosynthesis that is essential for survival of UV-induced DNA damage. Our study uncovers a role of H3K4me2 in coordinating the recovery of protein biosynthesis and homeostasis that is required for developmental growth and longevity after DNA damage.

2020-10-13 | PXD015354 | Pride

Global proteomics analysis reveals a role for histone variant H3.3 in the response to starvation

Project description:Periodic starvation of animals induces large shifts in metabolism, but may also influence many other cellular systems and can lead to adaption to prolonged starvation conditions. To date, there is limited understanding of how starvation affects gene expression, particularly at the protein level. Here, we have used mass spectrometry‐based quantitative proteomics to identify global changes in the C. elegans proteome due to acute starvation of young adult animals. Measuring changes in abundance of up to 7,000 proteins, we show that acute starvation rapidly alters the levels of hundreds of proteins, many involved in central metabolic pathways, highlighting key regulatory responses. Surprisingly, we also detect changes in the abundance of chromatin‐associated proteins including specific linker histones, histone variants and histone post‐translational modifications associated with the epigenetic control of gene expression. A null mutant for one of these proteins, the histone H3.3 variant HIS‐71, identified a subset of proteins whose abundance no longer varied in response to starvation. This mutant also displayed defects in starvation stress resistance and showed a reduced adult lifespan. To maximise community access to these data, they are presented in an online searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/).

2015-06-17 | PXD001723 | Pride

Mechanisms of stop codon readthrough mitigation reveal principles of GCN1 mediated translational quality control

Project description:Readthrough into the 3′ untranslated region (3′UTR) of the mRNA results in the production of aberrant proteins. Metazoans efficiently clear readthrough proteins, but the underlying mechanisms remain unknown. Here, we show in C. elegans and mammalian cells that readthrough proteins are targeted by a coupled, two-level quality control pathway involving the BAG6 chaperone complex and the ribosome collision-sensing protein GCN1. Readthrough proteins with hydrophobic C-terminal extensions are recognized by SGTA-BAG6 and ubiquitylated by RNF126 for proteasomal degradation. Additionally, cotranslational mRNA decay initiated by GCN1 and CCR4/NOT limits the accumulation of readthrough products. Unexpectedly, selective ribosome profiling uncovered a general role of GCN1 in regulating translation dynamics when ribosomes encounter nonoptimal codons, a feature of 3′UTR sequences. Dysfunction of GCN1 results in mRNA and proteome imbalance, increasingly perturbing transmembrane proteins and collagens during aging. These results define GCN1 as a key factor acting during translation in maintaining protein homeostasis.

2023-06-09 | PXD037037 | Pride

Transcription profiling of muscle from wild type and fer-1 mutant C. elegans

Project description:In vertebrate muscle, loss of Dysferlin results in the activation of compensatory muscle gene expression, even at pre-pathological stages. We hypothesized that if C. elegans fer-1 is also expressed in muscle, then fer-1 mutant worms might also exhibit compensatory muscle gene expression. To test this hypothesis, we used Affymetrix microarrays to profile gene expression from synchronized wild type and fer-1 mutant adults. To improve the specificity of this approach, we profiled two well characterized loss-of-function fer-1 mutants (hc1ts and hc24ts) and considered genes that changed similarly in both mutants as fer-1-regulated transcripts. Experiment Overall Design: For each genotype, six individual replicates were performed and were hybridized to Affymetrix C. elegans Genechips using the manufacturerâs recommended protocols. The best four preparations (as determined by the overall Pearson Correlation within a genotype) were used for RNA labeling and hybridization.

2009-07-05 | E-GEOD-16753 | biostudies-arrayexpress

Short-term mild temperature-stress-induced alterations in C. elegans phosphoproteome

Project description:Exposure to mild early-life stresses can slow down the aging process, in which protein phosphorylation might be an essential regulator. Currently, the understanding of phosphorylation-based regulatory networks under mild early-life stress remains elusive. Herein, we systematically analyzed the phosphoproteomes of C. elegans, which were treated with three kinds of mild temperature (15°C, 20°C, and 25°C) from two different short-term groups (10 min and 60 min). By utilizing iTRAQ-based quantitative phosphoproteomic approach, a total of 18187 phosphorylation sites from 3330 proteins were identified. Volcano plots illustrated that the phosphorylation abundance of 374 proteins (15°C) and 347 proteins (25°C), were significantly changed, respectively. Gene ontology, KEGG pathway and protein-protein interaction network analysis revealed that these phosphoproteins were mainly involved in metabolism, translation, development, and lifespan determination. Motif analysis of kinase substrates suggested that MAPK, CK and CAMK were most likely involved in the adaption processes. Moreover, 16 and 14 aging-regulated proteins underwent phosphorylation modifications under the mild stress of 15°C and 25°C, respectively, indicating these proteins might be important for maintaining long-term health. Further experiments of lifespan confirmed that the candidate phosphoproteins, e.g.; EGL-27, XNP-1, and GTBP-1 could regulate lifespan at 15°C, 20°C, and 25°C, and showed increased tolerance to heat and oxidative stresses. In summary, our findings provided a wealth of datasets to better understand the phosphorylation mechanism of mild early-life stresses in C. elegans.

2020-09-17 | PXD021081 | Pride

Conservation of mRNA and protein expression during development of C. elegans

Project description:Spatiotemporal control of gene expression is crucial for development and subject to evolutionary changes. Although proteins are the final product of most genes, the developmental proteome of an animal has not yet been comprehensively defined, and the correlation between RNA and protein abundance during development is largely unknown. Here, we globally measured and compared protein and mRNA expression changes during the life cycle of the nematodes C. elegans and C. briggsae, separated by ~30 million years of evolution. We observed that developmental mRNA and protein changes were highly conserved, to a surprisingly similar degree, but poorly correlated within a species, suggesting important and widespread post-transcriptional regulation. Post-transcriptional control was particularly well conserved if mRNA fold changes were buffered on the protein level, indicating a predominant repressive function. Finally, among divergently expressed genes, we identified insulin signaling, a pathway involved in life span determination, as a putative target of adaptive evolution. Samples of C. elegans and C. briggsae were collected at major developmental stages throughout the nematode life cycle. These stages comprise a population of mixed embryonic stages (E), populations of all four larval stages (L1, L2, L3, L4), late L4 larvae (LL4), young adults (YA), and a reference sample consisting of a mixture of all stages. To obtain synchronized worm populations, embryos were extracted by bleaching gravid adults and synchronized by starvation. Later stages were picked at fixed timepoints after determining the developmental stages by microscopic observation. For all stages, at least a single poly(A)-extracted mRNA library was sequenced on a single lane of an Illumina Genome Analyzer IIx.

2014-01-23 | E-GEOD-53359 | biostudies-arrayexpress

Differential modification of the C. elegans proteome in response to acute and chronic gamma radiation: link with reproduction decline

Project description:Ecosystems are chronically exposed to ionizing radiations. But environmental risk assessment of chronic exposure suffers from a lack of knowledge. Extrapolation of data from acute to chronic exposure is not always relevant, and can lead to uncertainties. In fact, effects could be different between the two irradiation modes, especially regarding reproduction endpoint, which is an ecologically relevant parameter . The free living nematode Caenorhabditis elegans is a particularly appropriate model organism to address this proteomic issues. With its fully sequenced genome and its short life cycle, C. elegans has been successfully used to study acute and chronic irradiation effects and their consequences on germline development and hatching. Results showed that a decrease of the number of progeny associated with a decrease of hatching success occurred from and above 30 Gy of acute irradiation.. In the present study, we decided to refine the understanding of molecular mechanisms of acute and chronic irradiation by a global proteome analysis. To do so, C. elegans were exposed to 3 common cumulated doses between acute and chronic exposure. These 3 doses, lower than the doses for which an effect on the reproduction was shown, were susceptible to allow us to find early and sensitive biomarkers of a reproduction decline. After exposure, global modification of the proteome expression was studied using a label free LC-MS/MS proteomic approach. Our objectives were to test the following hypotheses: (1) whether or not proteome expression varied with the dose, and with the irradiation mode; (2) if proteome expression modification was associated with effects on reproduction, with potential direct implications for ecological risk assessment.

2020-10-15 | PXD011731 | Pride

Transcripts regulated by elevated DKF-2 (WT background)

Project description:Transcriptional profiling was used to determine why DKF-2 (protein kinase D) deficiency and surfeit caused hypersensitivity and resistance, respectively, to a bacterial pathogen (P. aeruginosa 14). The studies show that DKF-2 contributes to C. elegans innate immunity by inducing expression of >75 genes that protect against pathogenic bacteria. Keywords: Induced imune response Three independent biological replicates were isolated for each strain. The strains for each replicate were grown and harvested in parallel.

2009-01-12 | E-GEOD-12456 | biostudies-arrayexpress

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