Unknown,Transcriptomics,Genomics,Proteomics

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RNA coimmunoprecipitations

ABSTRACT: Nascent polypeptides emerging from the ribosome during biogenesis can interact with many chaperones and protein homeostasis factors. The high sensitivity of RNA identification can be used to identify substrates of specific cotranslationally acting chaperones. Protein A-tagged (TAP-tag) chaperones associated with translating ribosomes were immunopurified by binding to magnetic IgG beads, washed, and chaperone complexes were eluted with TEV protease treatment. Immunopurified RNAs and total cell extract RNAs were isolated, reverse transcribed, coupled to Cy5 and Cy3 dyes, respectively, and comparatively hybridized to DNA microarrays Set of arrays that are part of repeated experiments

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Daniel Hogan

PROVIDER: E-GEOD-45794 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Defining the specificity of cotranslationally acting chaperones by systematic analysis of mRNAs associated with ribosome-nascent chain complexes.

del Alamo Marta M Hogan Daniel J DJ Pechmann Sebastian S Albanese Veronique V Brown Patrick O PO Frydman Judith J

PLoS biology 20110712 7

Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates thr ...[more]

PMID: 21765803

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Project description:Background MeCP2, methyl-CpG-binding protein 2, binds to methylated cytosines at CpG dinucleotides, as well as to unmethylated DNA, and affects chromatin condensation. MECP2 mutations in females lead to Rett syndrome, a neurological disorder characterized by developmental stagnation and regression, loss of purposeful hand use and speech, stereotypic hand movements, deceleration of brain growth, autonomic dysfunction and seizures. Most mutations occur de novo during spermatogenesis. Located at Xq28, MECP2 is subject to X inactivation, and affected females are mosaic. Rare hemizygous males suffer from a severe congenital encephalopathy. Methods To identify pathways mis-regulated by MeCP2 deficiency, microarray-based global gene expression studies were carried out on cerebellum of Mecp2 mutant mice. We compared transcript levels in mutant/wildtype male sibs of two different MeCP2-deficient mouse models at 2, 4 and 8 weeks of age. Increased transcript levels were evaluated by real-time quantitative RT-PCR. Chromatin immunoprecipitation assays were used to document in vivo MeCP2 binding to promoter regions of candidate target genes. Results In the mutants, several hundred genes showed altered expression levels. Twice as many were increased than decreased, and only 27 genes were differentially expressed at more than one time point. The number of misregulated genes was 30% lower in mice with an exon 3 deletion (Mecp2tm1.1Jae) than in mice with a larger deletion (Mecp2tm1.1Bird). Between the mutants, few misregulated genes overlapped at each time point. Real-time quantitative RT-PCR assays validated increased transcript levels for four genes: Irak1, interleukin-1 receptor-associated kinase 1; Fxyd1, phospholemman, associated with Na, K-ATPase; Reln, encoding reelin, an extracellular signaling molecule essential for neuronal lamination and synaptic plasticity; and Gtl2/Meg3, an imprinted maternally expressed non-translated RNA that serves as a host gene for C/D box snoRNAs and microRNAs. Chromatin immunoprecipitation assays documented in vivo MeCP2 binding to promoter regions of Fxyd1, Reln, and Gtl2. Conclusions Transcriptional profiling of cerebellum failed to detect significant global changes in Mecp2-mutant mice. Increased transcript levels of Irak1, Fxyd1, Reln, and Gtl2 may contribute to the neuronal dysfunction in MeCP2-deficient mice and individuals with Rett syndrome. Our data provide testable hypotheses for future studies of the regulatory or signaling pathways that these genes act on. A genetic modification design type is where an organism(s) has had genetic material removed, rearranged, mutagenized or added, such as knock out. Computed

Dataset Information

RNA coimmunoprecipitations

Publications

Defining the specificity of cotranslationally acting chaperones by systematic analysis of mRNAs associated with ribosome-nascent chain complexes.

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