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SRP046114
Mus musculus
2 Downloadable Samples
Illumina HiSeq 2500
Description
Nervous system (NS) development relies on coherent up-regulation of extensive sets of genes in a precise spatiotemporal manner. How such transcriptome-wide effects are orchestrated at the molecular level remains an open question. Here we show that 3'-untranslated regions (3'UTRs) of multiple neuronal transcripts contain A/U-rich cis-elements (AREs) recognized by tristetraprolin (TTP/Zfp36), an RNA-binding protein previously reported to destabilize mRNAs encoding predominantly cytokines, growth factors and proto-oncogenes. We further demonstrate that the efficiency of ARE-dependent mRNA degradation declines during neural differentiation due to a decrease in the TTP protein expression mediated by the NS-enriched microRNA miR-9. Our experiments with transgenenic cell lines suggest that TTP down-regulation is essential for proper neuronal differentiation. Moreover, inactivation of TTP in neuroblastoma cells or mouse embryonic fibroblasts induces major changes in their transcriptomes accompanied by significantly elevated expression of NS-specific genes. We conclude that the newly identified miR-9/TTP circuitry limits unscheduled accumulation of neuronal mRNAs in non-neuronal cells and ensures coordinated up-regulation of these transcripts in neurons. Overall design: 3''READS of undifferentiated and 3.5-day differentiated P19 cells
Publication Title
A post-transcriptional mechanism pacing expression of neural genes with precursor cell differentiation status.
Sample Metadata Fields
No sample metadata fields
View Samples- Saccharomyces cerevisiae
- 4 Downloadable Samples
- Illumina Genome Analyzer IIx
Description
The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNAP II) consists of repeated YSPTSPS heptapeptides and connects transcription with cotranscriptional events. Threonine-4 (Thr4) of the CTD repeats has been shown to function in histone mRNA 3'-end processing in chicken cells and in transcriptional elongation in human cells. Here, we demonstrate that, in budding yeast, Thr4, although dispensable for growth in rich media, is essential in phosphate-depleted or galactose-containing media. Thr4 is required to maintain repression of phosphate-regulated (PHO) genes under normal growth conditions and for full induction of PHO5 and the galactose-induced GAL1 and GAL7 genes. We identify genetic links between Thr4 and the histone variant Htz1 and show that Thr4, as well as the Ino80 chromatin remodeler, is required for activation-associated eviction of Htz1 specifically from promoters of the Thr4-dependent genes. Our study uncovers a connection between transcription and chromatin remodeling linked by Thr4 of the CTD. Overall design: RNA-seq of wild type and T4V mutant of budding yeast RNAP II CTD in duplicates
Publication Title
Threonine-4 of the budding yeast RNAP II CTD couples transcription with Htz1-mediated chromatin remodeling.
Sample Metadata Fields
Subject
View Samples- Mus musculus
- 1 Downloadable Sample
- Illumina Genome Analyzer II
Description
Genes containing multiple polyadenylation (polyA) sites express mRNA isoforms with variable 3' untranslated regions (3'UTRs). We found that short and long 3'UTR isoforms were relatively more abundant when genes were highly and lowly expressed, respectively, in human and mouse cells. Consistently, upregulated and downregulated genes were more likely to have shortened and lengthened 3'UTRs, respectively, when genes changed expression under different cell conditions or in response to extracellular stimuli. Using nuclear run-on assays, we found that RNA polymerase II (Pol II) was more likely to pause at the polyA site of highly expressed genes than that of lowly expressed ones. Moreover, in line with the difference in polyA site usage, highly expressed genes tend to have higher H3K4me3 and H3K36me3 levels but a lower density of nucleosome around promoter-proximal polyA sites relative to distal ones. Taken together, our results indicate that the efficiency of 3' end processing is generally coupled to transcriptional activity, leading to modulation of polyA site choice by transcription and thus connecting transcriptional control with post-transcriptional regulation via pre-mRNA processing. Overall design: Nascent RNA sequencing of C2C12 cell
Publication Title
Transcriptional activity regulates alternative cleavage and polyadenylation.
Sample Metadata Fields
No sample metadata fields
View Samples- Homo sapiens
- 4 Downloadable Samples
Affymetrix Human Exon 1.0 ST Array [probe set (exon) version (huex10st)
Description
Polyadenylation of mRNA precursors is mediated by a large multisubunit protein complex. Here we show that RBBP6 (retinoblastoma-binding protein 6), identified initially as an Rb- and p53-binding protein, is a component of this complex and functions in 3' processing in vitro and in vivo. RBBP6 associates with other core factors, and this interaction is mediated by an unusual ubiquitin-like domain, DWNN (domain with no name), that is required for 3' processing activity. The DWNN is also expressed, via alternative RNA processing, as a small single-domain protein (isoform 3 [iso3]). Importantly, we show that iso3, known to be down-regulated in several cancers, competes with RBBP6 for binding to the core machinery, thereby inhibiting 3' processing. Genome-wide analyses following RBBP6 knockdown revealed decreased transcript levels, especially of mRNAs with AU-rich 3' untranslated regions (UTRs) such as c-Fos and c-Jun, and increased usage of distal poly(A) sites. Our results implicate RBBP6 and iso3 as novel regulators of 3' processing, especially of RNAs with AU-rich 3' UTRs.
Publication Title
RBBP6 isoforms regulate the human polyadenylation machinery and modulate expression of mRNAs with AU-rich 3' UTRs.
Sample Metadata Fields
Cell line
View Samples- Mus musculus
- 66 Downloadable Samples
- Illumina HiSeq 2500, Illumina HiSeq 2000, Illumina Genome Analyzer II
Description
Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3' untranslated regions (3'UTRs) and/or coding sequences. How core cleavage and polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3'UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1, and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly influencing C/P events in 5' introns and U2 impacting those in efficiently spliced introns. Furthermore, PABPN1 regulates expression of transcripts with pAs near the transcription start site, a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results indicate that the abundance of different C/P factors and splicing factors plays diverse roles in APA, and is relevant to APA regulation in biological conditions. Overall design: knockdown experiments of 23 C/P factors, 3 splicing factors and U1D in mouse C2C12 myoblast cells
Publication Title
Systematic profiling of poly(A)+ transcripts modulated by core 3' end processing and splicing factors reveals regulatory rules of alternative cleavage and polyadenylation.
Sample Metadata Fields
No sample metadata fields
View Samples- Caenorhabditis elegans
- 2 Downloadable Samples
- Illumina Genome Analyzer IIx
Description
Caenorhabditis elegans is a major eukaryotic experimental system employed to unravel a broad range of cellular and biological processes. Despite the many advantages of C. elegans, biochemical approaches to study tissue-specific gene expression in postembryonic stages are challenging. Here we report a novel experimental approach that enables the efficient determination of tissue-enriched transcriptomes by rapidly releasing nuclei from major tissues of postembryonic animals followed by fluorescence-activated nuclei sorting (FANS). Furthermore, we developed and applied a deep sequencing method, named 3'end-seq, which is designed to examine gene expression and identify 3' ends of transcripts using a small quantity of input RNA. In agreement with intestinal specific gene expression, promoter elements of highly expressed genes are enriched for GATA elements and their functional properties are associated with processes that are characteristic for the intestine. In addition, we systematically mapped pre-mRNA cleavage and polyadenylation sites, or polyA sites, including >3,000 sites that have previously not been identified. The analysis of nuclear mRNA revealed widespread alternative polyA site use in intestinally expressed genes. We describe several novel approaches that will be of significance to the analysis of tissue specific gene expression using small quantity RNA samples from C. elegans and beyond. Overall design: 3'end-seq of transcriptomes for input and sorted nuclei
Publication Title
Analysis of C. elegans intestinal gene expression and polyadenylation by fluorescence-activated nuclei sorting and 3'-end-seq.
Sample Metadata Fields
Specimen part, Cell line, Subject
View Samples- Rattus norvegicus
- 12 Downloadable Samples
- Affymetrix Rat Gene 2.0 ST Array (ragene20st)
Description
Intravesical BCG Immunotherapy is the standard of care in treating non-muscle invasive bladder cancer, yet its mechanism of action remains elusive. Both innate and adaptive immune responses have been implicated in BCG activity. While prior research has indirectly demonstrated the importance of T cells and shown a rise in CD4+ T cells in bladder tissue after BCG, T cell subpopulations have not been fully characterized. We investigated the relationship between effector and regulatory T cells in an immune competent, clinically relevant rodent model of bladder cancer. Our data demonstrate that cancer progression in the MNU rat model of bladder cancer is characterized by a decline in the CD8/FoxP3 ratio, consistent with decreased adaptive immunity. By contrast, treatment with intravesical BCG leads to a large, transient rise in the CD4+ T cell population in the urothelium, and is both more effective and immunogenic compared to intravesical chemotherapy. Interestingly, whole transcriptome expression profiling of post-treatment intravesical CD4+ and CD8+ T cells revealed minimal differences in gene expression after BCG treatment. Together, our results suggest that while BCG induces T cell recruitment to the bladder, the T cell phenotype does not markedly change, implying that combining T cell activating agents with BCG might improve clinical activity.
Publication Title
Intravesical BCG Induces CD4<sup>+</sup> T-Cell Expansion in an Immune Competent Model of Bladder Cancer.
Sample Metadata Fields
Specimen part, Treatment
View Samples- Homo sapiens
- 2 Downloadable Samples
- Affymetrix Human Genome U133 Plus 2.0 Array (hgu133plus2)
Description
The composition of the matrix molecules is important in in vitro cell culture experiments of e.g. human cancer invasion and vessel formation. Currently, the mouse Engelbreth-Holm-Swarm (EHS) sarcoma -derived products, such as Matrigel, are the most commonly used tumor microenvironment mimicking (TMEM) matrices for experimental studies. However, since Matrigel is non-human in origin, its molecular composition does not accurately simulate human TMEM and we expect myogel to be more natural environment for human cancer cells. The environment may have crucial impact on cell behavior and gene expression.
Publication Title
A novel human leiomyoma tissue derived matrix for cell culture studies.
Sample Metadata Fields
Cell line
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