Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Chromatin immunoprecipitation of Sir-2.1 low copy transgenic C.elegans

ABSTRACT:

ORGANISM(S): Caenorhabditis elegans

SUBMITTER: Janos Demeter

PROVIDER: E-SMDB-3540 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

A role for SIR-2.1 regulation of ER stress response genes in determining C. elegans life span.

Viswanathan Mohan M Kim Stuart K SK Berdichevsky Ala A Guarente Leonard L

Developmental cell 20051101 5

C. elegans SIR-2.1, a member of the Sir-2 family of NAD(+)-dependent protein deacetylases, has been shown to regulate nematode aging via the insulin/IGF pathway transcription factor daf-16. Treatment of C. elegans with the small molecule resveratrol, however, extends life span in a manner fully dependent upon sir-2.1, but independent of daf-16. Microarray analysis of worms treated with resveratrol demonstrates the transcriptional induction of a family of genes encoding prion-like glutamine/aspar ...[more]

PMID: 16256736

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Project description:U2AF65 is an essential splicing factor involved in the 3'splice site recognition dureing the first steps of spliceosome assembly. In addition, this protein has nucleocytoplasmic shuttling activity and the Drosophila homologue has been implicated in mRNA export. We have used microarray hybridization coupled to RNA immunoprecipitation from HeLa cell cytoplasmic extracts using anti-U2AF65 antibodies to identify mRNA molecules associated with the U2AF65 splicing factor; Sample preparation methods (Gama-Carvalho et al, 2006, submitted); Suspension HeLa cells were grown in DMEM, 10% FCS, Pen/Strep and split 1:2 the day before harvesting. Post-nuclear cytoplasmic extracts were obtained as described (Herbert and Hecht 1999), using 5x107 cells/ml of lysis buffer. mRNAs were isolated by immunoprecipitation from 200ïl of precleared HeLa cell extract, using the anti-U2AF65 MC3 mAb (Gama-Carvalho, Krauss et al. 1997) or the Bb7 anti-PTB mAb (ATCC) for 2h at 4ÂºC. Immune complexes were precipitated with a slurry with 50% of protein A/protein G agarose beads (Amersham), blocked with 100ïg/ïl of tRNA and RNAse free BSA (Ambion), by rotating for 1h at 4ÂºC and washed with lysis buffer. Complexes bound to the beads were eluted with TES buffer (10mM Tris, 0.5M EDTA, 0.5% SDS, pH 8.0) by heating at 65ÂºC for 10 minutes and Trizol extracted. Polyadenilated RNA in the cell extract (input) and in the immunoprecipitation sample was reverse transcribed, end-tagged and PCR amplified using the Super SMART PCR cDNA synthesis kit from Clontech, following manufacturer instructions. 1ïg of input or precipitated RNA was used per RT-PCR reaction to produce samples for microarray hybridization. For each set of samples (input and immunoprecipitated) a 100ïl test PCR reaction was performed according to manufacturer instructions and analyzed by agarose gel electrophoresis to select ideal conditions for amplification in the linear range. PCR reactions were pooled to obtain enough material for microarray hybridization. 15ïg of PCR amplified cDNA from input and immunoprecipitated samples were fragmented, end labeled with biotin and hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays as described (Brodsky, Meyer et al. 2005), following standard Affymetrix hybridization protocols. Brodsky, A. S., C. A. Meyer, et al. (2005). "Genomic mapping of RNA polymerase II reveals sites of co-transcriptional regulation in human cells." Genome Biol 6(8): R64. Gama-Carvalho, M., R. D. Krauss, et al. (1997). "Targeting of U2AF65 to sites of active splicing in the nucleus." J Cell Biol 137(5): 975-87. Herbert, T. P. and N. B. Hecht (1999). "The mouse Y-box protein, MSY2, is associated with a kinase on non-polysomal mouse testicular mRNAs." Nucleic Acids Res 27(7): 1747-53. Experiment Overall Design: RNA immunoprecipitation was performed from HeLa cell extracts with anti-U2AF65 antibodies. For each immunoprecipitation experiment, two Affimetrix microarrays were hybridized with equal amount of total extract cDNA or cDNA from the immunoprecipitated sample. This series contains microarray data from three independant RNA immunoprecipitation experiments

Project description:PTB is multifunctional RNA binding protein reported to be involved in splicing, 3' -end processing, stability and translational regulation. Sample preparation methods (Gama-Carvalho et al, 2006, submitted); Suspension HeLa cells were grown in DMEM, 10% FCS, Pen/Strep and split 1:2 the day before harvesting. Post-nuclear cytoplasmic extracts were obtained as described (Herbert and Hecht 1999), using 5x107 cells/ml of lysis buffer. mRNAs were isolated by immunoprecipitation from 200ïl of precleared HeLa cell extract, using the anti-U2AF65 MC3 mAb (Gama-Carvalho, Krauss et al. 1997) or the Bb7 anti-PTB mAb (ATCC) for 2h at 4ÂºC. Immune complexes were precipitated with a slurry with 50% of protein A/protein G agarose beads (Amersham), blocked with 100ïg/ïl of tRNA and RNAse free BSA (Ambion), by rotating for 1h at 4ÂºC and washed with lysis buffer. Complexes bound to the beads were eluted with TES buffer (10mM Tris, 0.5M EDTA, 0.5% SDS, pH 8.0) by heating at 65ÂºC for 10 minutes and Trizol extracted. Polyadenilated RNA in the cell extract (input) and in the immunoprecipitation sample was reverse transcribed, end-tagged and PCR amplified using the Super SMART PCR cDNA synthesis kit from Clontech, following manufacturer instructions. 1ïg of input or precipitated RNA was used per RT-PCR reaction to produce samples for microarray hybridization. For each set of samples (input and immunoprecipitated) a 100ïl test PCR reaction was performed according to manufacturer instructions and analyzed by agarose gel electrophoresis to select ideal conditions for amplification in the linear range. PCR reactions were pooled to obtain enough material for microarray hybridization. 15ïg of PCR amplified cDNA from input and immunoprecipitated samples were fragmented, end labeled with biotin and hybridized to Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays as described (Brodsky, Meyer et al. 2005), following standard Affymetrix hybridization protocols. Brodsky, A. S., C. A. Meyer, et al. (2005). "Genomic mapping of RNA polymerase II reveals sites of co-transcriptional regulation in human cells." Genome Biol 6(8): R64. Gama-Carvalho, M., R. D. Krauss, et al. (1997). "Targeting of U2AF65 to sites of active splicing in the nucleus." J Cell Biol 137(5): 975-87. Herbert, T. P. and N. B. Hecht (1999). "The mouse Y-box protein, MSY2, is associated with a kinase on non-polysomal mouse testicular mRNAs." Nucleic Acids Res 27(7): 1747-53. Experiment Overall Design: We have used microarray hybridization coupled to RNA immunoprecipitation from HeLa cell cytoplasmic extracts using anti-PTB antibodies to identify mRNA molecules associated with the PTB splicing factor

Dataset Information

Chromatin immunoprecipitation of Sir-2.1 low copy transgenic C.elegans

Publications

A role for SIR-2.1 regulation of ER stress response genes in determining C. elegans life span.

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