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Temporal response of DCs to LPS stimulation: 4sU_sequencing

ABSTRACT: Regulation of RNA levels is critical for the response to external stimuli and determined through the interplay between RNA production, processing and degradation. Despite the centrality of these processes, most global studies of RNA regulation do not distinguish their separate contributions and relatively little is known about how they are temporally integrated. Here, we combine metabolic labeling of RNA with advanced RNA quantification assays and computational modeling to estimate RNA transcription and degradation during the response of immune dendritic cells (DCs) to pathogens, a critical and tightly regulated step in innate immunity. We find that transcription regulation plays a major role in shaping most temporal changes in RNA levels, but that changes in degradation rate are important for shaping sharp ‘peaked’ responses. We find that transcription changes precede corresponding RNA changes by a small lag (15-30 min), which is shorter for induced than for repressed genes. Massively parallel sequencing of the entire RNA population – including non-polyadenylated transcripts – allows us to estimate RNA processing, and identify specific groups of transcripts, mostly cytokines and transcription factors, undergoing enhanced mRNA maturation. This suggests an additional role for splicing in regulating mRNA maturation. Our method provides a new quantitative approach to study key steps in the integrative process of RNA regulation.

ORGANISM(S): Mus musculus

PROVIDER: GSE25432 | GEO | 2011/04/24

SECONDARY ACCESSION(S): PRJNA133787

REPOSITORIES: GEO

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Temporal response of DCs to LPS stimulation: 4sU_sequencing

Project description:Regulation of RNA levels is critical for the response to external stimuli and determined through the interplay between RNA production, processing and degradation. Despite the centrality of these processes, most global studies of RNA regulation do not distinguish their separate contributions and relatively little is known about how they are temporally integrated. Here, we combine metabolic labeling of RNA with advanced RNA quantification assays and computational modeling to estimate RNA transcription and degradation during the response of immune dendritic cells (DCs) to pathogens, a critical and tightly regulated step in innate immunity. We find that transcription regulation plays a major role in shaping most temporal changes in RNA levels, but that changes in degradation rate are important for shaping sharp M-bM-^@M-^XpeakedM-bM-^@M-^Y responses. We find that transcription changes precede corresponding RNA changes by a small lag (15-30 min), which is shorter for induced than for repressed genes. Massively parallel sequencing of the entire RNA population M-bM-^@M-^S including non-polyadenylated transcripts M-bM-^@M-^S allows us to estimate RNA processing, and identify specific groups of transcripts, mostly cytokines and transcription factors, undergoing enhanced mRNA maturation. This suggests an additional role for splicing in regulating mRNA maturation. Our method provides a new quantitative approach to study key steps in the integrative process of RNA regulation. Sequencing of 4sU-labeled RNA taken from a 7 samples time-series (one sample every 1 hour) during the response of DCs to LPS stimulation. 4-thiouridine was added 45 minutes prior to sample collection. Data presented here for six timepoints: 0, 1, 3-6 hrs. 2hr timepoint not included.

2011-04-24 | E-GEOD-25432 | biostudies-arrayexpress

Estimating RNA dynamics using one time point for one sample in a single-pulse metabolic experiment

Project description:Over the past decade, experimental procedures such as metabolic labeling for determining RNA turnover rates at the transcriptome-wide scale have been widely adopted. Several computational methods to estimate RNA processing and degradation rates from such experiments have been suggested, but they all require several RNA sequencing samples. Here we present a method that can estimate RNA processing and degradation rates from a single sample. To this end, we use the Zeisel model and take advantage of its analytical solution, reducing the problem to solving a univariate non-linear equation on a bounded domain. The approach is computationally rapid and enables inference of rates that correlate well with previously published datasets. In addition to saving experimental work and computational time, having a sample-based rate estimation has several advantages. It does not require an error-prone normalization across samples and enables the use of replicates to estimate uncertainty and perform quality control. Finally the method and theoretical results described here are general enough to be useful in other settings such as nucleotide conversion methods.

2020-05-12 | GSE150286 | GEO

Specific processing of meiosis related transcript is linked to final maturation in human oocytes

Project description:Human meiosis in oocytes entails a fine regulation of the transcriptome to support late oocyte growth and early embryo development, both key to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic passages, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 oocytes at different meiotic maturation stages, both in vivo ovulated and matured in vitro. We found abundant UTR processing, mostly at the 3’-, of meiosis-related genes between the stages of GV and MII, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very little differences among GV oocytes across several lengths of in vitro maturation, as long as they did not reach MII, suggesting a specific association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, though specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3’-UTR shortening, drives a selective translational regulation of transcripts necessary to reach the final meiotic maturation.

2023-05-27 | GSE213267 | GEO

Targeted protein degradation reveals Pol II heterogeneity and functional diversity

Project description:RNA polymerase II (Pol II) subunits are thought to be involved in various transcription-associated processes, but it is unclear whether they play different regulatory roles in modulating gene expression. Here, we performed nascent and mature transcript sequencing after the acute degradation of 12 mammalian Pol II subunits and profiled their genomic binding sites and protein interactomes to dissect their molecular functions. We found that Pol II subunits contribute differently to Pol II cellular localization and transcription process and preferentially regulate RNA processing (such as RNA splicing and 3’ end maturation). Genes sensitive to the depletion of different Pol II subunits tend to be involved in diverse biological functions and show different RNA half-lives. Sequences, associated protein factors, and RNA structures are correlated with Pol II subunit-mediated differential gene expression. These findings collectively suggest that the heterogeneity of Pol II and different genes appear to depend on some of the subunits.

2022-08-09 | PXD035866 | iProX

Seb1 proximity dependent biotinylation (PDB) in Schizosaccharomyces pombe

Project description:Transcription by RNA polymerase I (RNAPI) represents most of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal RNA (rRNA). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent pre-rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. Our project is to show that the conserved RNA-binding Seb1 is a pausing-promoting factor for RNA polymerases I to control cotranscriptional RNA processing. Here, we did a proximity dependent biotinylation followed by mass spectrometry (PDB-MS) of the Seb1 protein in order to assess for physical interactions with the RNAPI transcription machinery. A mutant E. coli BirA enzyme is fused to the Seb1 protein. This mutant version of BirA uses biotin to catalyze the formation of biotinoyl-5′-AMP (bioAMP), thereby generating a ‘cloud’ of activated biotin molecules that can react with free primary amines (most often lysine residues) of neighboring proteins. This experiment will support the conclusion that Seb1 is located at the rDNA locus.

2023-05-02 | PXD037768 | Pride

AUD Biomarkers Study (Proteomic and Genomic Analysis of Biospecimens)

Project description:Study purpose: to explore the entire spectrum of proteomic and genomic changes (amongst others) involved in diseases and in healthy/control populations. The Study is designed to discover biomarkers, develop and validate diagnostic assays, instruments and therapeutics as well as other medical research. Specifically, researchers may analyze proteins, RNA, DNA copy number changes, including large and small (1,000-100,000 kb) scale rearrangements, transcription profiles, epigenetic modifications, sequence variation, and sequence in both diseased tissue and case-matched germline DNA from Subjects.

| 62369 | ecrin-mdr-crc

Effect of LMP7 and MECL1-immunoproteasome subunits deficiency on the transcriptome of mouse bone marrow-derived dendritic cells

Project description:As regulators of protein degradation, proteasomes regulate practically all cellular functions. It is therefore logical to assume that replacement of the constitutive proteasome (CP) by its IFN- inducible homolog immunoproteasome (IP) could have far reaching effects on cell function. Accordingly, recent studies have revealed important roles for IPs in immune cells beyond MHC I-peptide processing. Moreover, the expression of IPs in non-immune cells from non-inflamed tissues suggests that the involvement of IPs is not limited to the immune system. We demonstrate here that IP-deficiency affects the transcription of 8104 genes in maturing dendritic cells (DCs). This occurs mainly through non-redundant regulation of key immune-related transcription factors by CPs and IPs. Additionally, IP-deficiency decreases DC's efficiency to activate CD8+ T cells in vivo. Our study reveals that the broad cellular roles of IPs could rely on transcription regulation and, more importantly, illustrates how IP-deficiency could generate MHC I-peptide processing-independent phenotypes. Examination of the transcriptome of WT and immunoproteasome-deficient cells at 4 different time points of dendritic cell maturation, in 4 experimental replicates (total of 32 samples).

2014-07-02 | E-GEOD-52616 | biostudies-arrayexpress

ChIP seq: STAT1 and STAT3 transcription factors sense and interpret changes in inflammation through engagement of specific enhancers

Project description:Using ChIP-Seq analysis we explore regulation of gene expression changes during inflammatory peritonitis induced by challenge with bacterial ligands administered via the intra-peritoneal route. A resuspended lypholised supernatant of Staphylococcus epidermidis (SES) was administered with or without ex vivo generated CD4+ T-cells (Th1-polarised) to understand the role of adaptive immune responses in shaping inflammatory processes. To study the role of Signal transducer and Activator of Transcription 1 and 3 (STAT1/STAT3) we performed ChIP-seq on peritoneal membrane sections isolated from wt and Il-6 knockout mice three hours post-challenge. Membrane sections were snap frozen in liquid nitrogen prior to downstream processing using established ChIP-seq protocols (Illumina Truseq).

2022-04-04 | E-MTAB-10758 | biostudies-arrayexpress

The conserved RNA-binding protein Seb1 promotes cotranscriptional ribosomal RNA processing by controlling RNA polymerase I progression

Project description:Transcription by RNA polymerase I (RNAPI) represents the majority of the transcriptional activity in eukaryotic cells and is associated with the production of mature ribosomal rRNAs (rRNAs). As several rRNA maturation steps are coupled to RNAPI transcription, the rate of RNAPI elongation directly influences processing of nascent rRNA, and changes in RNAPI transcription rate can result in alternative rRNA processing pathways in response to growth conditions and stress. However, factors and mechanisms that control RNAPI progression by influencing transcription elongation rate remain poorly understood. We show here that the conserved fission yeast RNA-binding protein Seb1 associates with the RNAPI transcription machinery and promotes RNAPI pausing states along the rDNA. The overall faster progression of RNAPI at the rDNA in Seb1-deficient cells impaired cotranscriptional rRNA processing and the production of mature rRNAs. Given that Seb1 also influences pre-mRNA processing by modulating RNAPII progression, our findings unveil Seb1 as a pausing-promoting factor for RNA polymerases I and II to control cotranscriptional RNA processing.

2023-05-02 | GSE212930 | GEO

A multi-omics dataset of heat-shock response in the yeast RNA binding protein Mip6 (NMR assay)

Project description:<p>Gene expression is a biological process regulated at different molecular levels, including chromatin accessibility, transcription, and RNA maturation and transport. In addition, these regulatory mechanisms have strong links with cellular metabolism. Here we present a multi-omics dataset that captures different aspects of this multi-layered process in yeast. We obtained RNA-seq, metabolomics, and H4K12Ac ChIP-seq data for wild-type and mip6delta strains during a heat-shock time course. Mip6 is an RNA-binding protein that contributes to RNA export during environmental stress and is informative of the contribution of post-transcriptional regulation to control cellular adaptations to environmental changes. The experiment was performed in quadruplicate, and the different omics measurements were obtained from the same biological samples, which facilitates the integration and analysis of data using covariance-based methods. We validate our dataset by showing that ChIP-seq, RNA-seq and metabolomics signals recapitulate existing knowledge about the response of ribosomal genes and the contribution of trehalose metabolism to heat stress.</p>

2020-02-14 | MTBLS1320 | MetaboLights

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