ABSTRACT: 3'UTR sequencing was performed in indicated C.elegans strains to obtain information about alterations in 3'UTR lengths and alternative polyadenylation events.
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.
Project description:We applied a middle-down proteomics strategy for large scale protein analysis during in vivo development of Caenorhabditis elegans. We characterized post-translational modifications (PTMs) on histone H3 N-terminal tails at eight time points during the C. elegans lifecycle, including embryo, larval stages (L1 to L4), dauer and L1/L4 post dauer. Histones were analyzed by our optimized middle-down protein sequencing platform using high mass accuracy tandem mass spectrometry. This allows quantification of intact histone tails and detailed characterization of distinct histone tails carrying co-occurring PTMs. We measured temporally distinct combinatorial PTM profiles during C. elegans development. We show that the doubly modified form H3K23me3K27me3, which is rare or non-existent in mammals, is the most abundant PTM in all stages of C. elegans lifecycle. The abundance of H3K23me3 increased during development and it was mutually exclusive of the active marks H3K18ac, R26me1 and R40me1, suggesting a role for H3K23me3 in to silent chromatin. We observed distinct PTM profiles for normal L1 larvae and for L1-post dauer larvae, or L4 and L4 post-dauer, suggesting that histone PTMs mediate an epigenetic memory that is transmitted during dauer formation. Collectively, our data describe the dynamics of histone H3 combinatorial code during C. elegans lifecycle and demonstrate the feasibility of using middle-down proteomics to study in vivo development of multicellular organisms.
Project description:Recent work has shown that small non-coding RNAs, including miRNAs, serve an important role in controlling gene expression during development and disease. However, little detailed information exists concerning the relative expression patterns of small RNAs during development of C. elegans. Here we use recent advances in high-throughput sequencing technology to show that expression of non-coding small RNAs, including miRNAs, changes dynamically during development and in the different sexes of C. elegans; approximately 16% of known miRNAs changed over 10 fold in expression during C. elegans development and about 12% of miRNAs showed major changes in expression between males and hermaphrodites of C. elegans. These results should lead to a better understanding of the expression and function of small RNAs in C. elegans development. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf Examination of small RNA expression in six different developmental stages of hermaphrodites (Embryo, mid-L1, mid-L2, mid-L3, mid-L4, young adult), and young adult males (dpy-28;him-8) and spermatogenesis-defective young adult hermaphrodites (spe-9). The number of sequence reads for miRNA was assessed from the raw sequence data from Solexa sequencing using perfect sequence matching to known miRNAs (miRBase Release 11.0).
Project description:Effective toxicological testing of the vast number of new and existing chemicals currently in use will require efficient and cost effective methods. We evaluated the utility of a simple, low cost toxicity testing system employing the nematode Caenorhabditis elegans to identify toxicologically relevant changes in gene expression. The objective of this research is to determine genomic and proteomic responses in the model nematode C. elegans to exposures to representatives of several classes of toxic industrial chemicals/materials (TICs/TIMs). A total of 3 chemicals (acrylamide, cadmium chloride, and mercuric chloride) were used in these experiments. Affymetrix GeneChip for C. elegans was used to examine genome-wide responses in the 19,000+ genes of this model organism. Mid-vulval L4 worms were exposed to one of four concentrations of acrylamide, cadmium or mercury for 4h and 8h. Exposures were performed in triplicate.
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.
Project description:AMPK (AAK-2) and calcineurin (TAX-6) mediate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC (CREB regulated transcriptional coactivator). We performed microarrays to examine the transcriptional responses elicited by the pro-longevity: activation of AMPK, deactivation of calcineurin, and decrease of CREB (CRH-1) activity. Gene expression profiles for crh1 (nn3315) and tax-6 (ok2065) mutants, aak-2c (aa1-321) overexpressers and WT (N2) controls were obtained by measuring RNA levels in replicate pools of 3000 synchronized L4 worms. Three replicate pools of each strain were prepared on separate days.
Project description:We aimed at identifying the transcriptomes of single and double mutants of the two C.elegans Mi2 homologues, let-418 and chd-3. Embryos depleted for one or both genes were manually sorted and synchronised at the 24- and 100-cell stages, and their transcriptome compared to control embryos in order to identify de-regulated target genes, and determine whether the two Mi2 proteins are functionally redundant.
Project description:Transcriptome profiling of Lir3 C.elegans mutants. Larval stage L4 worms were used for RNA isolation from N2 (Bristol), Q40 (polyglutamine model) genetic background; Lir3 and wiltype controls. Experiments were done in triplicates using ribosomal RNA depletion. Libraries were sequenced with 50bp reads on Illumina HiSeq2500 platform
Project description:In nature, animals often face feast or famine conditions. We aimed to identify the miRNAs of Caenorhabditis elegans that changed their expression under starvation conditions in stage L4 larvae. Our results highlight 14 miRNAs that show differential expression in starved versus well-fed larvae. In particular, miRNAs of the miR-35-3p/miR-41-3p family were upregulated 6-20 fold upon starvation. We verified the upregulation of miR-35-3p with qPCR. Additionally, we showed that the expression of gld-1, important in ovogenesis, and a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was higher. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans. Illumina small RNA sequencing of starved and well-fed L4 worms.
Project description:Hemes are essential but potentially cytotoxic cofactors that participate in critical and diverse biological processes. Although the pathway and intermediates for heme biosynthesis have been well defined, the intracellular networks which mediate heme trafficking remain unknown. Caenorhabditis elegans and related helminths are natural heme auxotrophs requiring environmental heme for growth and development. We exploited this auxotrophy to identify HRG-1 and HRG-4 in C. elegans and show that they are essential for heme homeostasis and normal vertebrate development. We demonstrate that heme deficiency upregulates expression of hrg-4 and its evolutionarily conserved paralog hrg-1. Depletion of either HRG-1 or HRG-4 in worms results in disruption of organismal heme sensing and abnormal response to heme analogs. HRG-1 and HRG-4 are novel transmembrane proteins that bind heme and have evolutionarily conserved functions. Transient knockdown of hrg-1 in zebrafish leads to hydrocephalus, yolk tube malformations, and, most strikingly, profound defects in erythropoiesis - phenotypes that are fully rescued by worm HRG-1. These findings reveal unanticipated and conserved pathways for cellular heme trafficking in animals that defines the paradigm for eukaryotic heme transport. Uncovering the mechanisms of heme transport in C. elegans will provide novel insights into human disorders of heme metabolism and generate unique anthelmintics to combat worm infestations. Experiment Overall Design: As a first-step toward understanding heme homeostasis at the molecular level, we performed genome-wide microarrays to identify genes that are transcriptionally regulated by heme. For microarray analysis, synchronized F2 larvae were re-inoculated in mCeHR-2 medium supplemented with 4, 20 or 500 uM hemin and harvested at the late L4 stage for mRNA prep and probe hybridization to Affymetrix C. elegans Whole Genome Expression Arrays. Total RNA from three biological replicates were used at each hemin concentration. Data from worms grown in mCeHR-2 medium with 4 and 500 uM hemin were compared to data from worms grown in 20 uM hemin. Microarray data were verified with Microarray Suite 5.0 (Affymetrix) and Robust Multichip Average Method (RMA, R package). Results from MAS 5.0 and RMA analyses provided with 375 genes that showed 1.6 fold change in 4 and 500 uM hemin when compared to data from 20 uM hemin samples. Statistical analyses identified changes in 375 genes from worms grown in either 4 or 500 µM heme (see Methods). The microarray results were validated by qRT-PCR (Supplementary Fig. 1) and the 375 heme-responsive genes were classified into eight categories based on their relative changes in gene expression (Table I). The data from the microarray study show that �1.9 % of genes in the worm genome are transcriptionally responsive to heme. Notably, of the 375 genes, 164 had some sequence identity in human genome databases at the amino acid level, and >90 % of the genes had no ascribed function in the C.