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SRP066173
Mus musculus
8 Downloadable Samples
Illumina HiSeq 2000
Description
The underlying relation between Parkinson disease (PD) etiopathology and its major risk factor, aging, is largely unknown. The nature of the specific age-related mechanisms promoting PD onset is experimentally difficult to elucidate because aging is a highly complex process contributed by multiple factors. Recent evidence, however, established a strong and causative link between genome stability and aging. To investigate a possible nexus between DNA damage accumulation, aging, and PD we examined DNA repair pathways associated with aging in laboratory animal models and human cases. We demonstrate that dermal fibroblasts from PD patients display flawed nucleotide excision repair (NER) capacity and that NER-defective mice exhibit typical PD-like pathological alterations, including decreased dopaminergic innervation in the striatum, increased phospho-synuclein levels, and defects in mitochondrial respiration. NER mouse mutants are also more sensitive to the prototypical PD toxin MPTP and their transcriptomic landscape shares important similarities with that of PD patients. Overall, our results demonstrate that specific defects in DNA repair impact the dopaminergic system, are associated with human PD pathology, and might therefore constitute a novel risk factor for PD by affecting the aging process. Overall design: In total 8 samples were analyzed, 4 controls and 4 Ercc1 mutants.
Publication Title
Inefficient DNA Repair Is an Aging-Related Modifier of Parkinson's Disease.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Caenorhabditis elegans
- 4 Downloadable Samples
- Illumina Genome Analyzer II
Description
Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes. Overall design: Arrested L1 worms were grown in liquid culture supplemented with 2mM 4SU or 6SG. 250,000 worms were sufficient for one iPAR-CLIP experiment. Living adult worms were transferred to NGM plates and crosslinked on ice using a Stratalinker (Stratagene) with customized 365nm UV-lamps (energy setting: 2J/cm2). Worms were lysed on ice by douncing in NP40 lysis buffer (50 mM HEPES-K pH 7.5, 150 mM KCl, 2 mM EDTA, 0.5% (v/v) NP-40, 0.5 mM DTT, protease inhibitor cocktail (Roche)). Cleared lysates were treated with RNase T1 (Fermentas) (final concentration 1U/?l) for 15 min at 22ºC. GLD-1::GFP::FLAG fusion proteins were immunoprecipitated for 1h at 4ºC using anti-FLAG antibody (Sigma, F3165) coupled to Protein G magnetic beads (Invitrogen). For one iPAR-CLIP experiment (1ml cleared lysate obtained from 250,000 worms), 300µl beads and 150µg antibody were used. Immunoprecipitates were treated with RNase T1 (100U/?l) for exactly 12 min at 22 ºC. Subsequently, PAR-CLIP was carried out as described previously (Hafner et al, 2010). cDNA libraries were sequenced on a Genome Analyzer II (Illumina).
Publication Title
In vivo and transcriptome-wide identification of RNA binding protein target sites.
Sample Metadata Fields
Cell line, Subject
View Samples- Caenorhabditis elegans
- 2 Downloadable Samples
- Illumina HiSeq 2000
Description
Animal mRNAs are regulated by hundreds of RNA binding proteins (RBPs). The identification of RBP targets is crucial for understanding their function. A recent method, PAR-CLIP, uses photoreactive nucleosides to crosslink RBPs to target RNAs in cells prior to immunoprecipitation. Here, we establish iPAR-CLIP (in vivo PAR-CLIP) to determine, at nucleotide resolution, transcriptome-wide target sites of GLD-1, a conserved, germline-specific translational repressor in C. elegans. We identified 439 reproducible targets and demonstrate an excellent dynamic range of target detection by iPAR-CLIP. Upon GLD-1 knock-down, protein but not mRNA expression of the 439 targets was specifically and highly significantly upregulated, demonstrating functionality. Finally, we discovered strongly conserved GLD-1 binding sites nearby the start codon of target genes. We propose that GLD-1 interacts with the translation machinery nearby the start codon, a so far unknown mode of gene regulation in eukaryotes. Overall design: PolyA mRNA was extracted from young adult wildtype (N2) worms and young adult germline less worms (glp-4(bn2) TS) to identify and quantify genes expressed in the young adult germline by sequencing. 2x100 paired end sequencing was performed according to the protocol on the Illumina HiSeq 2000.
Publication Title
In vivo and transcriptome-wide identification of RNA binding protein target sites.
Sample Metadata Fields
Cell line, Subject
View Samples- Homo sapiens
- 12 Downloadable Samples
- IlluminaHiSeq2500
Description
RNA helicases are important regulators of gene expression that act by remodeling RNA secondary structures and as RNA-protein interactions. Here, we demonstrate that MOV10 has an ATP-dependent 5'' to 3'' in vitro RNA unwinding activity and determine the RNA-binding sites of MOV10 and its helicase mutants using PAR-CLIP. We find that MOV10 predominantly binds to 3'' UTRs upstream of regions predicted to form local secondary structures and provide evidence that MOV10 helicase mutants are impaired in their ability to translocate 5'' to 3'' on their mRNA targets. MOV10 interacts with UPF1, the key component of the nonsense-mediated mRNA decay pathway. PAR-CLIP of UPF1 reveals that MOV10 and UPF1 bind to RNA in close proximity. Knockdown of MOV10 resulted in increased mRNA half-lives of MOV10-bound as well as UPF1-regulated transcripts, suggesting that MOV10 functions in UPF1-mediated mRNA degradation as an RNA clearance factor to resolve structures and displace proteins from 3'' UTRs. Overall design: Flp-In T-REx HEK293 cells expressing FLAG/HA-tagged MOV10 WT, MOV10 K530A, MOV10 D645N and UPF1 were used to determine the protein-RNA interaction sites of RNA helicases MOV10 and UPF1 as well as MOV10 inactive variants using PAR-CLIP in combination with next generation sequencing. mRNA half-life changes of MOV10-targeted mRNA were determined by measuring mRNA half-lives by mRNA sequencing of mock and MOV10-depleted HEK293 cells.
Publication Title
MOV10 Is a 5' to 3' RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3' UTRs.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 6 Downloadable Samples
- Illumina HiSeq 2000
Description
The conserved human LIN28 RNA-binding proteins function in development, maintenance of pluripotency and oncogenesis. We used PAR-CLIP and a newly developed variant of this method, iDo-PAR-CLIP, to identify LIN28B targets as well as sites bound by the individual RNA binding domains of LIN28B in the human transcriptome at nucleotide resolution. The position of target binding sites reflected the known structural relative orientation of individual LIN28B binding domains, validating iDo-PAR-CLIP. Our data suggest that LIN28B directly interacts with most expressed mRNAs and members of the let-7 microRNA family. The Lin28 binding motif detected in pre-let-7 was enriched in mRNA sequences bound by LIN28B. Upon LIN28B knock down, cell proliferation and the cell cycle were strongly impaired. Quantitative shotgun proteomics of LIN28B depleted cells revealed significant reduction of protein synthesis from its RNA targets that function in translation, mRNA splicing and cell cycle control. Computational analyses provided evidence that the strength of protein synthesis reduction correlated with the location of LIN28B binding sites within target transcripts. Overall design: We used PAR-CLIP and a newly developed variant of this method, iDo-PAR-CLIP, to identify LIN28B targets as well as sites bound by the individual RNA binding domains of LIN28B in the human transcriptome at nucleotide resolution.
Publication Title
Identification of LIN28B-bound mRNAs reveals features of target recognition and regulation.
Sample Metadata Fields
Cell line, Subject
View Samples- Homo sapiens
- 12 Downloadable Samples
- IlluminaHiSeq2000
Description
The RNA-binding protein RC3H1 (also known as ROQUIN) promotes TNFalpha mRNA decay via a 3''UTR constitutive decay element (CDE). Here, we applied PAR-CLIP to human RC3H1 to identify about 3800 mRNA targets with more than 16000 binding sites. A large number of sites are distinct from the consensus CDE and revealed a structure-sequence motif with U-rich sequences embedded in hairpins. RC3H1 binds preferentially short-lived and DNA damage induced mRNAs, indicating a role of this RNA-binding protein in the posttranscriptional regulation of the DNA damage response. Intriguingly, RC3H1 affects expression of NF-kB pathway regulators such as IkBalpha and A20. RC3H1 uses roquin and Zn-finger domains to contact a binding site in the A20 3''UTR, demonstrating a not yet recognized mode of RC3H1 binding. Knockdown of RC3H1 resulted in increased A20 protein expression, thereby interfering with IkB kinase and NF-kB activities, demonstrating that RC3H1 can modulate the activity of the IKK/NF-kB pathway. Overall design: We measured global mRNA decay rates in mock and RC3H1/RC3H2-depleted HEK293 cells. Transcription was blocked by Actinomycin D zero, one or two hours before harvesting. Total RNA was isolated in two biological replicates and subjected to polyA selection followed by high-throughput sequencing.
Publication Title
RC3H1 post-transcriptionally regulates A20 mRNA and modulates the activity of the IKK/NF-κB pathway.
Sample Metadata Fields
No sample metadata fields
View Samples- Mus musculus
- 6 Downloadable Samples
Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
The Wnt/alpha-catenin pathway plays a central role in epidermal homeostasis and regeneration but how it affects fibroblast fate decisions is unknown. Here, we investigated the effect of targeted alpha-catenin stabilization in dermal fibroblasts. Comparative gene expression profiling of Sca1- and Sca1+ neonatal fibroblasts, from upper and lower dermis respectively, confirmed that Sca1+ cells had a pre-adipocyte signature and revealed differential expression of Wnt/alphacatenin-associated genes. By targeting all fibroblasts or selectively targeting Dlk1+ lower dermal fibroblasts, we found that -catenin stabilization between E16.5 and P2 resulted in a reduction in the dermal adipocyte layer with a corresponding increase in dermal fibrosis and an altered hair cycle. The fibrotic phenotype correlated with a reduction in the potential of Sca1+ fibroblasts to undergo adipogenic differentiation ex vivo. Our findings indicate that Wnt/alpha-catenin signaling controls adipogenic cell fate within the lower dermis, which potentially contributes to the pathogenesis of fibrotic skin diseases.
Publication Title
β-Catenin Stabilization in Skin Fibroblasts Causes Fibrotic Lesions by Preventing Adipocyte Differentiation of the Reticular Dermis.
Sample Metadata Fields
Specimen part
View Samples- Sus scrofa
- 6 Downloadable Samples
- Affymetrix Porcine Genome Array (porcine)
Description
Calcific aortic valvular disease (CAVD) is characterized by sclerosis of the aortic valve leaflets and recent clinical studies have linked several other risk factors to this disease, including male sex. In this study we examined potential sex-related differences in gene expression profiles between porcine male and female valvular interstitial cells (VICs) to explore possible differences in CAVD propensity on the cellular level.
Publication Title
Sex-related differences in gene expression by porcine aortic valvular interstitial cells.
Sample Metadata Fields
Sex, Specimen part
View Samples- Homo sapiens
- 157 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
Information about the genes that are preferentially expressed during the course of Alzheimers disease (AD) could improve our understanding of the molecular mechanisms involved in the pathogenesis of this common cause of cognitive impairment in older persons, provide new opportunities in the diagnosis, early detection, and tracking of this disorder, and provide novel targets for the discovery of interventions to treat and prevent this disorder. Information about the genes that are preferentially expressed in relationship to normal neurological aging could provide new information about the molecular mechanisms that are involved in normal age-related cognitive decline and a host of age-related neurological disorders, and they could provide novel targets for the discovery of interventions to mitigate some of these deleterious effects.
Publication Title
Gene expression profiles in anatomically and functionally distinct regions of the normal aged human brain.
Sample Metadata Fields
Sex, Age, Specimen part, Disease, Disease stage, Race
View Samples- Mus musculus
- 9 Downloadable Samples
- Affymetrix Mouse Genome 430 2.0 Array (mouse4302)
Description
Background & Aims: MiRNAs are small (~22 nucleotide), non-coding RNA molecules that regulate gene expression through imperfect complementarity with target messenger RNAs. The function of miRNA in mammalian organogenesis is largely unknown. Conditional loss-of-function of Dicer, the enzyme that processes precursor miRNA transcripts into their mature, active form, has been shown to cause severe defects in a number of organ systems. Here we address the role of Dicer in liver development and function. Methods: Mice lacking Dicer function in hepatocytes were generated using an Afp-Cre strain to drive deletion of a floxed Dicer allele. Deletion of the flox-dicer allele was confirmed by quantitative PCR. Decreased miRNA levels detected by quantitative RT-PCR and in situ hybridization confirmed loss of Dicer function. Gene expression microarray analysis was performed on liver RNA from P28 mutant and control mice. Liver sections from mutant and control mice ranging from embryonic stages through 3-4 months of age were examined and liver function tests were performed on adult mice. Results: Mice lacking hepatocyte Dicer function were born alive at the expected frequency, and had grossly normal appearance and behavior. Despite the loss of mature miRNA, hepatic function was normal, as reflected by normal blood gludose, albumin, cholesterol, and bilirubin. However, mutant mice between 2-4 months of age exhibit progressive hepatocyte damage, elevated ALT/AST, with evidence of balanced proliferation and apoptosis in the lobule. Microarray analysis indicates large-scale changes in gene expression, with increased expression of many miRNA targets, as well as imprinted genes. Conclusions: Loss of miRNA processing in the liver at late gestation has a remarkably mild phenotype, suggesting that miRNAs do not play an essential role in hepatic physiology. However, miRNA deficiency results in hepatocyte apoptosis and balanced hepatocyte regeneration. Finally, microarray analysis of gene expression in mutant liver suggests a previously unrecognized role for Dicer in the repression of imprinted genes.
Publication Title
Hepatic function is preserved in the absence of mature microRNAs.
Sample Metadata Fields
Age, Specimen part
View Samples