RNA-seq from animals treated with RNAi against prp-8, prp-6, and prp-31
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ABSTRACT: Transcriptome analysis of a population of control animals and RNAi-treated to partially inactivate genes that are homologs to human genes causing Retinitis Pigmentosa. RNA-seq analyses were performed at L3 larval stage in wild type, and smg-1(r861) mutants that have a defective Non-mediated decay pathway. RNA-seq experiments were also performed in adults glp-4(bn2) mutants that lack of germline. synchronized N2 and smg-1(r861) L1 larvae fed for 24 hours at 20 °C with the RNAi clones of prp-6, prp-8, prp-31 and gfp; and 5-day adult glp-4(bn2) worms grown at 25 °C and fed first, for 72 hours with OP50 and next, for 48 more hours with the RNAi clones of prp-8, prp-31 and gfp
Project description:Transcriptome analysis of a population of control animals and RNAi-treated to partially inactivate genes that are homologs to human genes causing Retinitis Pigmentosa. RNA-seq analyses were performed at L3 larval stage in wild type, and smg-1(r861) mutants that have a defective Non-mediated decay pathway. RNA-seq experiments were also performed in adults glp-4(bn2) mutants that lack of germline.
Project description:RNA-seq of C.elegans strain N2 (wt) with and without sfa-1 RNAi at adult day 3 and day 15, C. elegans strain DA1116 (eat-2(ad1116)) with and without sfa-1 RNAi at adult day 3, day 15, and day 27, C. elegans strain SS104 (glp-4(bn2)) with and without hrp-2 RNAi at adult day 1, and HeLa cells with and without SF1 siRNA.
Project description:To determine mRNA expression levels glp-4(bn2); let-7(wild-type) and glp-4(bn2); let-7(mn112) animals, RNA was purified from total worms collected at late L4 stage using TRI Reagent (MRC).
Project description:All eukaryotes studied to date have the capacity to detect and degrade mRNAs harboring premature translation termination codons (PTCs) in a process called nonsense-mediated mRNA decay (NMD) (reviewed in Wagner E & Lykke-Andersen J, 2002). This surveillance system allows the cell to prevent the expression of potentially harmful truncated proteins. trans-acting factors required for NMD in Drosophila are the proteins UPF1, UPF2, UPF3, SMG-1, SMG-5 and SMG-6. To identify mRNAs naturally regulated by NMD, we analyzed expression profiles in Drosophila cells RNAi-depleted of known NMD factors using high-density oligonucleotide arrays.
Project description:At times, it can be difficult to discern if a lack of overlap in reported interactions for a protein-of-interest reflects differences in methodology or biology. A case in point is the prion protein (PrP) which is best known for its central role in prion disorders. Despite having over two dozen interactors reported, there is little consensus regarding their importance for understanding the biology of PrP. In such instances, systematic analyses of protein-protein networks across diverse paradigms can provide valuable insights. Here, we interrogated the PrP interactome in four distinct mouse cell lines. Analyses made use of identical affinity capture reagents and sample processing workflows. To enable the discrimination of specific from non-specific binders, negative controls were generated from PrP knockout lines of the respective cell models, and the relative levels of peptides were quantified with the help of isobaric labels. The study uncovered 26 proteins, which reside in proximity to PrP. All of these proteins are predicted to have access to the outer face of the plasma membrane, and approximately half of them were not reported to interact with PrP before. Strikingly, although several proteins exhibited profound co-enrichment with PrP in a given model, except for Ncam1, no protein was highly enriched in all four PrP-specific interactome datasets.
Project description:Tendon is a hypocellular tissue that contains functional cable-like units of type I collagen responsible for the transmission of force from muscle to bone. In the setting of injury or disease, patients can develop chronic tendinopathies that are characterized by pain, loss of function and persistent inflammatory changes that are often difficult to treat. Platelet-rich plasma (PRP) has shown promise in the treatment of chronic tendinopathy, but little is known about the mechanisms by which PRP can improve tendon healing. PRP contains many different growth factors and cytokines, and since these proteins can both activate and inhibit various signaling pathways it has been challenging to determine precisely which signaling pathways and cellular responses are most important. Using state-of-the-art bioinformatics tools and genome wide-expression profiling, the purpose of this study was to determine the signaling pathways activated within cultured tendon fibroblasts in response to PRP treatment. Tendon fibroblasts were isolated from rat tail tendons and embedded in 3D type I collagen gels. Cells were treated with PRP or PPP for 24 hours, and total RNA was extracted for hybridization on Affymetrix arrays.
Project description:Analysis of small RNAs with 5'p, 3'OH Small RNAs (18-32nt) expressed in purified male sperm, hermaphrodite oocytes, embryos, N2, glp-4(bn2), and eri-1(mg366) were cloned and sequenced by 454 and Illumina high-throughput sequencing.
Project description:The molecular function of the cellular prion protein (PrPC) and the mechanism by which it may contribute to neurotoxicity in prion diseases and Alzheimer’s disease (AD) are only partially understood. Mouse neuroblastoma Neuro2a cells and, more recently, C2C12 myocytes and myotubes have emerged as popular models for investigating the cellular biology of PrP. Mouse epithelial NMuMG cells might become attractive models for studying the possible involvement of PrP in a morphogenetic program underlying epithelial-to-mesenchymal transitions. Here we describe the generation of PrP knockout clones from these cell lines using CRISPR-Cas9 knockout technology. More specifically, knockout clones were generated with two separate gRNAs targeting recognition sites on opposite strands within the first hundred nucleotides of the Prnp coding sequence. Several PrP knockout clones were isolated and genomic insertions and deletions near the CRISPR-target sites were characterized. Subsequently, deep quantitative global proteome analyses that recorded the relative abundance of > 3000 proteins were undertaken to begin to characterize the molecular consequences of PrP deficiency. The levels of ~120 proteins were shown to reproducibly correlate with the presence or absence of PrP, with most of these proteins as belonging to extracellular components, cell junctions or the cytoskeleton.
Project description:The glp-1/NOTCH pathway is a conserved pathway that plays an important role in developmental control. To study the effect of natural genetic variation on perturbations in this pathway, we used N2xCB4856 recombinant inbred lines of the nematode Caenorhabditis elegans. These were treated with empty vector or gld-1 RNAi, where gld-1 is a key developmental gene in the glp-1/NOTCH pathway. The recombinant inbred lines were exposed for two generation to the treatment and 47 hour old L4 juveniles were collected for RNA isolation. In total 39 RILs were exposed to the empty-vector treatment and 46 RILs were exposed to the gld-1 treatment. Gene expression was quantified using microarrays.