Years after the discovery that Dicer is a key enzyme in gene-silencing, the role of its helicase domain remains enigmatic. Here we show that this domain is critical for accumulation of certain endogenous small interfering RNAs (endo-siRNAs) in C. elegans. The domain is required for the production of the direct products of Dicer, or primary endo-siRNAs, and consequently, affects levels of downstream intermediates, the secondary endo-siRNAs. Consistent with the role of endo-siRNAs in silencing, their loss correlates with an increase in cognate mRNA levels. We find that the helicase domain of Dicer is not required for microRNA (miRNA) processing, or RNA interference following exposure to exogenous double-stranded RNA. Comparisons of wildtype and helicase-defective strains using deep-sequencing analyses show that the helicase domain is required by a subset of annotated endo-siRNAs, in particular, those associated with the slightly longer 26 nucleotide small RNA species containing a 5' guanosine. Overall design: We reintroduced either wildtype Dicer, or Dicer harboring a mutation (K39A) in it''s helicase domain, into dcr-1(ok247) mutant worms via transgene rescue. We then used high-throughput sequencing to compare levels of small RNAs present in each of these strains.
Dicer's helicase domain is required for accumulation of some, but not all, C. elegans endogenous siRNAs.
Cell line, Subject
View SamplesmRNA expression in Eµ-Myc lymphoma cells expressing or lacking miR-17~92 Overall design: Eµ-Myc B-cell lymphomas harboring conditional alleles of miR-17~92 were cultured with or without 4-OHT to generate isogenic tumour cells with homozygous deletion of miR-17~92. Wild type (fl) and miR-17~92-deleted (del) Eµ-Myc cells were cultured for 48 hours under regular growth conditions, and RNA isolated for sequencing analysis.
The miR-17∼92 microRNA Cluster Is a Global Regulator of Tumor Metabolism.
Specimen part, Subject
View SamplesWe describe here an interrupted reprogramming strategy to generate "induced Progenitor-Like (iPL) cells" from Alveolar Epithelial Type II (AEC-II) cells. A carefully defined period of transient expression of reprogramming factors (Oct4, Sox2, Klf4 and c-Myc; OSKM) is able to rescue the limited in vitro clonogenic capacity of AEC-II cells, potentially by activation of a bipotential progenitor-like state.
Interrupted reprogramming of alveolar type II cells induces progenitor-like cells that ameliorate pulmonary fibrosis.
Specimen part
View SamplesComparison of the changes in mitochondrial gene expression of cells in which extracellular growth factors and/or mitogens have been added.
Extracellular growth factors and mitogens cooperate to drive mitochondrial biogenesis.
Specimen part
View SamplesAnalysis of a SigX knockout mutant of Pseudomonas aeruginosa H103 strain in LB.
The absence of SigX results in impaired carbon metabolism and membrane fluidity in Pseudomonas aeruginosa.
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View SamplesThe gene expression profile of blood drawn from healthy individuals was studied over a period of six months, at five time points. The gene expression profiles appeared to be constant over one month and to slightly vary over three months. A small proportion of genes were found to be differentially regulated according to gender. Differential gene regulation by age (in subjects 2555 years of age versus subjects > 55 years of age) was not observed.
A longitudinal study of gene expression in healthy individuals.
Sex, Age, Specimen part, Subject
View SamplesThe intestinal epithelium is continuously renewed by a pool of intestinal stem cells expressing Lgr5. We show that deletion of the key autophagy gene Atg7 affects the survival of Lgr5+ intestinal stem cells. Mechanistically, this involves defective DNA repair, oxidative stress, and altered interactions with the microbiota. This study highlights the importance of autophagy in maintaining the integrity of intestinal stem cells.
Essential role for autophagy protein ATG7 in the maintenance of intestinal stem cell integrity.
Specimen part
View SamplesEffect of LUBEL catalytic dead mutation in immune response Overall design: Mutation was introduced in the LUBEL catalytic region by CRISPR/Cas9 techonology in Drosophila melanogaster and their transcriptome was compared in control (sample 23930 to 23941) and e.coli pricked samples (sample 28984 to 28995).
Linear ubiquitination by LUBEL has a role in Drosophila heat stress response.
Sex, Specimen part, Subject
View SamplesEffect of LUBEL catalytic dead mutation upon Heastshock Overall design: Mutation was introduced in CG11321 catalytic region by CRISPR/Cas9 techonology in Drosophila melanogaster and transcriptome was compared in untreated and heatshocked samples
Linear ubiquitination by LUBEL has a role in Drosophila heat stress response.
Treatment, Subject
View SamplesThe cytosolic protein Sharpin is as a component of the linear ubiquitin chain assembly complex (LUBAC), which regulates NF-B signaling in response to specific ligands. Its inactivating mutation in Cpdm (chronic proliferative dermatitis mutation) mice causes multi-organ inflammation, yet this phenotype is not transferable into wildtype mice by hematopoietic stem cell transfer. Recent evidence demonstrated that Cpdm mice additionally display low bone mass, but the cellular and molecular causes of this phenotype remained to be established. Here we have applied non-decalcified histology together with cellular and dynamic histomorphometry to perform a thorough skeletal phenotyping of Cpdm mice. We show that Cpdm mice display trabecular and cortical osteopenia, solely explained by impaired bone formation, whereas osteoclastogenesis is unaffected. We additionally found that Cpdm mice display a severe disturbance of articular cartilage integrity in the absence of joint inflammation, supporting the concept that Sharpin-deficiency affects mesenchymal cell differentiation. Consistently, Cpdm mesenchymal cells displayed reduced osteogenic capacitiy ex vivo, yet this defect was not associated with impaired NF-B signaling. A molecular comparison of wildtype and Cpdm bone marrow cell populations further revealed that Cpdm mesenchymal cells produce higher levels of Cxcl5 and lower levels of IL1ra. Collectively, our data demonstrate that skeletal defects of Cpdm mice are not caused by chronic inflammation, but that Sharpin is as a critical regulator of mesenchymal cell differentiation and gene expression. They additionally provide an alternative molecular explanation for the inflammatory phenotype of Cpdm mice and the absence of disease transfer by hematopoetic stem cell transplantation.
Sharpin Controls Osteogenic Differentiation of Mesenchymal Bone Marrow Cells.
Specimen part
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