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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of S. cerevisiae to identify Upf1p-associated transcripts in S. cerevisiae

ABSTRACT: The goal of this experiment was to identify transcripts associated with the S. cerevisiae Upf1 protein. Experiment Overall Design: The RNA population from four independent pairs of Upf1p-TAP and mock affinity selections were analyzed by using Affymetrix Yeast Genome S98 Arrays.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Marcus Johansson

PROVIDER: E-GEOD-9486 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Association of yeast Upf1p with direct substrates of the NMD pathway.

Johansson Marcus J O MJ He Feng F Spatrick Phyllis P Li Chunfang C Jacobson Allan A

Proceedings of the National Academy of Sciences of the United States of America 20071217 52

Nonsense-mediated mRNA decay (NMD) is a surveillance mechanism that detects and degrades transcripts containing premature translation termination codons. Gene expression profiling experiments have shown that inactivation of the NMD pathway leads to the accumulation of both aberrant, nonsense-containing mRNAs, and many apparently wild-type transcripts. Such increases in transcript steady-state levels could arise from direct changes in the respective mRNA half-lives, or indirectly, as a consequenc ...[more]

PMID: 18087042

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Microarray analysis of TAP::ALG-1 associated RNAs isolated from synchronized 'wild-type' animals and 'mir-58' mutants

Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein (C. elegans strain WS4303). We crossed the TAP::ALG-1 transgene into the mir-58(n4640) mutant background to generate the strain WS5041. For simplicity, we will hereafter term the TAP::ALG-1 transgenic animals as wild typeand the transgenic WS5041 animals as mir-58. We compared the mRNA population that coimmunopurified with TAP::ALG-1 from synchronized L4 stage wild-type animals with that from synchronized L4 stage mir-58 mutant animals by one-color Affymetrix gene arrays. miR-58 target mRNAs should be specifically underrepresented in the latter samples. Strains WS4303 (wt) and WS5041 (mir-58) were used for TAP::ALG-1 IPs. All experiments were conducted in three independent replicates. For each replicate, WS4303 and WS5041 were grown in parallel. 150 ng of TAP::ALG-1 associated RNA isolated from synchronized late L4 animals were sent to the GeneCore facilty in Heidelberg, Germany (http://www.genecore.embl.de/index.cfm), and the microarray data were generated according to their standard protocol (Weinmann et al. 2009).

2012-03-01 | E-GEOD-32942 | biostudies-arrayexpress

Purification of TAP::ALG-1 complexes and associated RNA from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2) animals

Project description:To discover new miRNA targets, we generated a C. elegans transgenic line expressing a functional N-terminally Tandem Affinity Purification (TAP) tagged ALG-1 protein We purified TAP::ALG-1 complexes from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2, serving as a mock control) animals and hybridized the associated mRNAs to two-color microarrays RNA isolated from WS4303 and N2 (mock control) animals was analyzed for each IP sample and for each total worm extract sample. Three independent biological replicates have been performed. Long-oligo whole-genome C. elegans arrays, produced by the Genome Sequencing Center at Washington University in St. Louis (http://genome.wustl.edu/genome/celegans/microarray/ma_gen_info.cgi), were used for analysis. Log2 ratios (immunopurified RNA over total RNA from extract) were calculated for each gene. Microarrays generated from WS4303 animals were compared to microarrays generated from N2 mock control animals by a Student’s two sample t test.

2012-03-01 | E-GEOD-32941 | biostudies-arrayexpress

ChIP-chip of Hmt1-9myc from S. cerevisiae

Project description:A comprehensive genome-wide binding profile for Hmt1 was obtained through the method of ChIP-chip using NimbleGen high-resolution tiling microarrays. Of the approximately 1000 Hmt1-binding sites found, the majority fall within or proximal to an ORF. However, Hmt1 occupancy is found at a number of other genomic features such as tRNA and snoRNA genes, thereby implicating a regulatory role in the biogenesis of these non-coding RNAs. RNA hybridization analysis shows that Hmt1 loss-of-function mutants display higher steady-state tRNA abundance relative to the wild-type. Co-immunoprecipitation studies show that Hmt1 interacts with the TFIIIB component Bdp1, suggesting a role for Hmt1 in modulating RNA pol III transcription to regulate tRNA production. ChIP-chip of Hmt1-9myc using Nimblegen high-resoluion tiling arrays

2012-12-25 | E-GEOD-40505 | biostudies-arrayexpress

Identification of miRNA target genes in C. elegans by RIP-chip-SRM

Project description:RIP-chip-SRM : a New Combinatorial Large Scale Approach Identifies a Set of Translationally Regulated bantam/miR 58 Targets in C. elegans RNA binding protein immunopurification + microarray + targeted protein quantification via Selected Reaction Monitoring This SuperSeries is composed of the following subset Series: GSE32941: Purification of TAP::ALG-1 complexes and associated RNA from mixed-stage TAP::ALG-1 transgenic (WS4303) and wild-type (N2) animals GSE32942: Microarray analysis of TAP::ALG-1 associated RNAs isolated from synchronized 'wild-type' animals and 'mir-58' mutants GSE32943: Expression analysis of WS4303 (wild type) vs WS5041 (mir-58 mutants) total mRNA Refer to individual Series

2012-03-01 | E-GEOD-32944 | biostudies-arrayexpress

Identification and functional characterization of proteins interacting with cohesin complex in yeast Saccharomyces cerevisiae.

Project description:To ensure equal separation of DNA, sister chromatids are held together from S phase to metaphase–anaphase transition by a multiprotein complex called cohesin. This makes it possible to establish chromosome biorientation, counteracts the pulling force of mitotic spindle microtubules, preventing premature sister chromatid separation, and ensures precise segregation of sister DNAs into daughter cells In order to better understand how the sister chromatid cohesion process is regulated we looked for new cohesin interacors. We constructed a yeast strain endogenously expressing TAP-tagged Scc1 (Scc1-TAP). Next, we performed a single-step TAP purification using an untagged strain (mock sample) or a strain expressing Scc1-TAP, followed by identification of co-purifying proteins by MS.

2023-10-24 | PXD037034 | Pride

A Novel HDAC Complex in the Control of Transcription and Genome Stability

Project description:The acetylation state of histones, controlled by histone acetyltransferases and deacetylases, profoundly affects DNA transcription and repair by modulating chromatin accessibility to the cellular machinery. The Schizosaccharomyces pombe HDAC Clr6 (human HDAC1) binds to different sets of proteins that define functionally distinct complexes: I, IM-bM-^@M-^Y and II. Here we determine the composition, architecture and functions of a new Clr6 HDAC complex, I'', delineated by the novel proteins Nts1, Mug165 and Png3. Deletion of nts1 causes increased sensitivity to genotoxins and deregulated expression of Tf2 elements, long non-coding RNA, subtelomeric and stress-related genes. Similar, but more pervasive, phenotypes are observed upon Clr6 inactivation, supporting the designation of complex I'' as a mediator of a key subset of Clr6 functions. We also reveal that with the exception of Tf2 elements, Clr6 I''-regulated loci and its genome-wide loading sites do not correlate. Instead, Nts1 loads at genes that are expressed in mid-meiosis, following oxidative stress, or are periodically expressed. Collective data suggest that Clr6 I'' has (i) indirect effects on gene expression, conceivably by mediating higher-order chromatin organization of sub-telomeres and Tf2 elements, and (ii) direct effects on the transcription of specific genes in response to certain cellular or environmental stimuli. Identification of the genome binindg sites for Nts1 (SPCC24B10.19C) in Schizosaccharomyces pombe

2014-07-10 | E-GEOD-57040 | biostudies-arrayexpress

Shg1p associated RNAs

Project description:RNA co-immunopurification with TAP-tagged Shg1p (a component of the Set1 histone methyltransferase complex, SET1C) from Saccharomyces cerevisiae. An untagged (BY4741) served as a control (Gerber et al. 2004). Cells were grown to mid-log phase in rich media and harvested by centrifugation. TAP-tagged Shg1 was affinity-purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input) and from the purified protein sample with the RNAeasy Kit (Qiagen). RNAs were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast oligo microarrays over night at 42 degrees in formamide-based hybridization buffer. For the method see experimental procedure for RNA IP (Gerber/Dichtl). An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.

2010-06-24 | E-GEOD-15247 | biostudies-arrayexpress

Reactivity of commercial antibodies targeting human histone H2B N-terminal tail on the Intel array platform

Project description:The aim of the study was to test the Intel array platform and map the H2B epitopes bound by commercial antibody reagents. We also tested the ability of SET7 PKMT to methylate Intel array peptides. We diluted antibody reagents 1:1000 and incubated dilutions on the silicon-based Intel array platorm. We mapped antibody binding epitopes and SET7 PKMT methylation target peptides using a novel, silicon-based array platform with 8820 individual peptide features comprising every possible contiguous peptide from 1-21 a.a. in length within the 21-mer N-terminal tail of human histone H2B.

2011-12-02 | E-GEOD-34034 | biostudies-arrayexpress

The fission yeast MTREC complex targets CUTs and unspliced mRNAs to the nuclear exosome

Project description:Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome. However, the mechanism by which they are recognized and targeted to the exosome is not fully understood. Here, we report that the MTREC complex, which has recently been shown to promote degradation of meiotic mRNAs and regulatory ncRNAs, is also the major nuclear exosome targeting complex for CUTs and unspliced pre-mRNAs in Schizosaccharomyces pombe. MTREC complex specifically binds to CUTs, meiotic mRNAs and unspliced pre-mRNA transcripts and targets these RNAs for degradation by the nuclear exosome, while the TRAMP complex has only a minor role in this process. The MTREC complex physically interacts with the nuclear exosome and with various RNA-binding and -processing complexes, coupling RNA processing to the RNA degradation machinery. Our study reveals the central role of the evolutionarily conserved MTREC complex in RNA quality control, and in the recognition and elimination of CUTs.

2015-04-22 | PXD001908 | Pride

Transcription profiling of two transgenic models familial hypertrophic cardiomyopathy mice vs wild type and non transgenic controls using pooled and non pooled heart extracts to identify differences in gene expression in FHC

Project description:Familial hypertrophic cardiomyopathy (FHC) is a disease characterized by ventricular hypertrophy, fibrosis, and aberrant systolic and/or diastolic function. We previously developed two transgenic mouse models that carry FHC associated mutations in alpha-tropomyosin (TM): FHC alpha-TM175 mice show patchy areas of mild ventricular disorganization and limited hypertrophy; whereas FHC alpha-TM180 mice exhibit severe hypertrophy and fibrosis and die within 6 months. To obtain a better understanding of the molecular mechanisms associated with the early onset of cardiac hypertrophy, we conducted a detailed comparative analysis of gene expression in 2.5-month-old control and FHC alpha-TM175 and alpha-TM180 ventricular tissue. Results show that 754 genes (from a total of 22,600) were differentially expressed between the NTG and the FHC hearts. There are 178 differentially regulated genes between NTG and the FHC alpha-TM175 hearts, 388 genes are differentially expressed between NTG and FHC alpha-TM180 hearts, and 266 genes are differentially expressed between FHC alpha-TM175 and FHC alpha-TM180 hearts. Genes that exhibit the largest increase in expression belong to the secreted/extracellular matrix category, and those with the most significant decrease in expression are associated with metabolic enzymes. Samples used for hybridization consisted of pooled (P) and non-pooled (NP) RNA extracts from the three groups namely NTG, alpha-TM175 and alpha-TM180. Ten hybridization experiments were performed in which each genotypic group was represented by two or more non-pooled (NP) individual RNA extracts and one pooled (P) sample that resulted from combining 20 individual heart extracts

2007-11-23 | E-GEOD-4678 | biostudies-arrayexpress

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