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SRP077288
Homo sapiens
24 Downloadable Samples
Illumina HiSeq 2000, NextSeq 500
Description
FAM46C is one of the most frequently mutated genes in multiple myeloma (MM) and encodes a protein of unknown function. Using a combination of in vitro and in vivo approaches, we demonstrate that FAM46C encodes an active cytoplasmic non-canonical poly(A) polymerase, which enhances mRNA stability and gene expression. Moreover, we also found that the reintroduction of active FAM46C into MM cell lines, but not its catalytically-inactive mutant, leads to broad polyadenylation and stabilization of mRNAs strongly enriched with those encoding endoplasmic reticulum-targeted proteins and induced cell death. This is, to our knowledge, the first report that directly associates cytoplasmic poly(A) polymerase with carcinogenesis. Furthermore, our data suggest that the human genome encodes at least eleven non-canonical poly(A) polymerases with four FAM46 family members. Since FAM46 proteins are differentially expressed during development, these proteins may positively regulate transcript stability and translational rate in a tissue-specific manner. Overall design: The H929 and SKMM1 MM cells were transduced with lentiviruses carrying FAM46CWTGFP (WT) or FAM46CD90A,D92AGFP (catalitic mutant). 72h after transgene delivery total RNA was extracted and RNA-seq libraries were prepared.
Publication Title
The non-canonical poly(A) polymerase FAM46C acts as an onco-suppressor in multiple myeloma.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Homo sapiens
- 21 Downloadable Samples
- IlluminaHiSeq2000
Description
A multi-subunit exosome complex is a major eukaryotic exoribonuclease that in the cytoplasm requires the SKI complex for activity. In yeast, SKI forms a heterotetramer and delivers RNA substrates directly into the exosome channel. Such cooperation requires Ski7 protein, which links the exosome and SKI complexes. However, since the human genome does not encode an orthologue of the yeast Ski7, the factor mediating SKI and exosome linkage in human cells is unknown. Proteomic analysis revealed that the human cytoplasmic exosome interacts with HBS1LV3, a protein encoded by a newly discovered short splicing isoform of HBS1L. HBS1LV3 recruits the SKI complex to the exosome. In contrast, the canonical HBS1L variant, HBS1LV1, acting as a ribosome dissociation factor, does not associate with the exosome and instead interacts with the mRNA surveillance factor PELOTA. HBS1LV3 contains a new domain of unknown structure with the short linear motif RxxxFxxxL, which is responsible for exosome binding, and may interact with the exosome core subunit RRP43 in way that resembles the association between Rrp6 RNase and Rrp43 in yeast. Depletion of HBS1LV3 and the SKI complex helicase SKI2W similarly affected the transcriptome by strongly upregulating a large number of genes. Moreover, following HBS1LV3 or SKI2W depletion the half-lives of representative upregulated mRNAs were increased, thus supporting the involvement of HBS1LV3 and SKI2W in the same mRNA degradation pathway. In contrast, HBS1LV1 depletion had little effect on transcriptome homeostasis. Our data indicate that human HBS1LV3 is the long-sought factor that links the exosome and SKI complexes to regulate cytoplasmic mRNA decay. Overall design: Examination of siRNA-mediated silencing in HEK293 cell lines. To identify transcripts that are degraded by cytoplasmic SKI/HBS1LV3/exosome supercomplexes, we used specific siRNAs to knock down HBS1LV1, HBS1LV3 or SKIV2L gene expression in (i) WT HEK293 cells and (ii) HEK293 cells rescued with siRNA insensitive protein. Analyses were performed in triplicate.
Publication Title
A short splicing isoform of HBS1L links the cytoplasmic exosome and SKI complexes in humans.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 6 Downloadable Samples
- NextSeq 500
Description
We analysed the effect of depriving the human cell of the catalytic activity of the nuclear 5' to 3' exoribonuclease XRN2. Catalytic amino acids in this protein had been defined previously, so it was possible to design a mutated catalytically inactive form of the protein (XRN2D233A-D235A) (PMID: 19194460). We created 293 Flp-In T-REx stable cell lines that induciby silence endogenous XRN2, and concomitantly express wild-type or inactive XRN2 in fusion with EGFP at the C-terminus. Thus, complementation of silencing of endogenous XRN2 with the expression of mutant version of the protein allows to directly link potential phenotypes with the lack of XRN2 enzymatic activity. To this end we isolated total RNA from tetracycline-treated cells, depleted it from rRNA and conducted strand-specific deep sequencing. Overall design: 6 samples were analysed. 3 replicates of control cells (endogenous copy of XRN2 gene is silenced and catalytically active exogenous XRN2-EGFP is expressed) and 3 replicates of cells deprived of XRN2 ribonucleolytic activity (endogenous copy of XRN2 gene is silenced and catalytically inactive exogenous XRN2(D233AD235A)-EGFP is expressed)
Publication Title
Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system.
Sample Metadata Fields
Specimen part, Subject
View Samples- Macaca mulatta
- 10 Downloadable Samples
Affymetrix Rhesus Macaque Genome Array (rhesus)
Description
The majority of transplanted organs are recovered from deceased donors after brain death (BD). BD has been hypothesized to compromise organ quality in part from the activation of systemic inflammation. The objective of this study was to characterize the immune response induced by BD in a well controlled non-human primate (NHP) model. Assessment of physiologic parameters (blood pressure, heart rate, urinary output, catecholamines, and cerebral angiograms) was used to confirm BD. After 6h of BD, we monitored changes in the peripheral blood by flow cytometry, liver gene expression by microarray and liver protein expression by Western blotting and immunohistochemistry (IHC). BD was indicated by a rapid increase in blood pressure followed by hemodynamic instability, hypotension, diabetes insipidus and the absence of cerebral blood flow and brain stem reflexes. Within the peripheral blood IL-6 levels and neutrophils increased and myeloid dendritic cells decreased in BD NHP when compared to living donor controls. Genes related to innate inflammatory response and apoptosis were significantly upregulated in BD NHP. BD livers showed increased expression of suppressor of cytokine signaling 3 (SOCS3) protein and the danger associated molecular pattern protein S100A9. Increased expression of intracellular cellular adhesion molecule 1 (ICAM-1) and major histocompatibility complex (MHC) II, neutrophil accumulation, and products of oxidative stress (carboxy methyl lysine (CML) and hydroxynonenal (HNE)) were detected by IHC in livers. Conclusion: These data indicate that BD leads to the rapid activation of an inflammatory response within the liver involving components of the innate immune response at the gene and protein levels. The activation of these inflammatory pathways may provide one explanation for the reduced post-transplant function of organs from brain dead donors.
Publication Title
Early activation of the inflammatory response in the liver of brain-dead non-human primates.
Sample Metadata Fields
Sex, Age, Specimen part
View Samples- Homo sapiens
- 3 Downloadable Samples
- Illumina Genome Analyzer II
Description
In adult cancers, epigenetic changes and aberrant splicing of the DNMT3B is commonly observed, and the pattern of gene methylation and expression has been shown to be modified by DNMT3B7, a truncated protein of DNMT3B. Much less is known about the mechanism of epigenetic changes in the pediatric cancer neuroblastoma. To investigate if aberrant DNMT3B transcripts alter DNA methylation, gene expression and tumor phenotype in neuroblastoma, we measured DNMT3B isoform expression in primary tumors and cell lines. Higher levels of DNMT3B7 were detected in differentiated ganglioneuroblastomas compared to undifferentiated neuroblastomas, suggesting that expression of DNMT3B7 may induce a less clinically aggressive tumor phenotype. To test this hypothesis, we investigated the effects of forced DNMT3B7 in neuroblastoma cells. We found that DNMT3B7 expression significantly inhibited neuroblastoma cell proliferation in vitro, and in neuroblastoma xenografts, DNMT3B7 decreased angiogenesis and tumor growth. DNMT3B7-positive cells had higher levels of total genomic methylation, and RNA-sequencing revealed a dramatic decrease in expression of FOS and JUN family members, AP1 complex components. Consistent with the established antagonistic relationship between AP1 expression and retinoic acid receptor activity, decreased proliferation and increased differentiation was seen in the DNMT3B7-expressing neuroblastoma cells following treatment with all trans retinoic acid (ATRA) compared to controls. Our results demonstrate that high levels of DNMT3B7 modify the epigenome in neuroblastoma cells, induce changes in gene expression, inhibit tumor growth, and increase sensitivity to ATRA. Further knowledge regarding mechanisms by which DNMT3B7 regulates gene methylation may ultimately lead to the development of therapeutic strategies that reverse the epigenetic aberrations that drive neuroblastoma pathogenesis. Overall design: DNMT3B7, a truncated DNMT3B isoform, was stably transfected into an N-type neuroblastoma cell line (LA1-55n) using a Tet-off inducible system. DNMT3B7 expressing cells were compared to vector control cells after 21 days of induction.
Publication Title
Truncated DNMT3B isoform DNMT3B7 suppresses growth, induces differentiation, and alters DNA methylation in human neuroblastoma.
Sample Metadata Fields
Cell line, Subject
View Samples- Mus musculus
- 24 Downloadable Samples
- Illumina MouseWG-6 v2.0 R2 expression beadchip
Description
We derived a transcriptional signature of oncogenic KRAS by using the KF508 murine pancreatic ductal cell line with an inducible Lox-Stop-Lox (LSL) cassette in front of the KRASG12D oncogene to regulate transcription. This dataset allowed us to study the differential expression profile after oncogenic KRAS induction in mouse.
Publication Title
Master Regulators of Oncogenic KRAS Response in Pancreatic Cancer: An Integrative Network Biology Analysis.
Sample Metadata Fields
Cell line, Treatment
View Samples- Mus musculus
- 1 Downloadable Sample
- Illumina MouseWG-6 v2.0 expression beadchip
Description
This SuperSeries is composed of the SubSeries listed below.
Publication Title
Global transcriptome and chromatin occupancy analysis reveal the short isoform of GATA1 is deficient for erythroid specification and gene expression.
Sample Metadata Fields
Specimen part, Cell line
View Samples- Mus musculus
- 1 Downloadable Sample
- Illumina MouseWG-6 v2.0 expression beadchip
Description
The transcriptional activiy of GATA1s was compared to GATA1 through gene expression analysis in a cell line model with both erythroid and megakaryocyte differentiation.
Publication Title
Global transcriptome and chromatin occupancy analysis reveal the short isoform of GATA1 is deficient for erythroid specification and gene expression.
Sample Metadata Fields
Specimen part, Cell line
View Samples