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Affinity purification of ribosomes and associated RNAs from stress-treated cells

ABSTRACT: In this study, we systematically identified ribosome associated RNAs. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a, expressed under control of its native promoter, was affinity purified from whole cell extracts of cultures grown to mid-log phase. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. To analyze how changes in steady-state mRNA levels (= transcriptome) are related to changes of respective messages in the translatome, total RNA and ribosome associated RNA from stress-treated and untreated cells were analyzed with yeast cDNA microarrays. To achieve this, fluorescently labeled (Cy5) cDNA was prepared from total RNA isolated from yeast cell extracts and from affinity-purified ribosomes, and each sample was mixed with differentially labeled (Cy3) cDNA prepared from a common reference RNA pool, and competitively hybridized on yeast cDNA microarrays. We performed five independent experiments with untreated cells grown in minimal medium (t=0 reference) and three or more independent biological replicates of treated cells. Altogether, we sampled the cell's response to five different "stress treatments" that were applied for relatively short periods of time (10 or 20 minutes) to avoid secondary effects triggered by transcriptional adaption. A stimulus or stress experiment design type is where that tests response of an organism(s) to stress/stimulus. e.g. osmotic stress, behavioral treatment Time: Duration of the treatment Growth Condition: Type of treatment

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE13653 | GEO | 2009/05/12

SECONDARY ACCESSION(S): PRJNA113893

REPOSITORIES: GEO

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Affinity purification of ribosomes and associated RNAs from stress-treated cells

Project description:In this study, we systematically identified ribosome associated RNAs. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a, expressed under control of its native promoter, was affinity purified from whole cell extracts of cultures grown to mid-log phase. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. To analyze how changes in steady-state mRNA levels (= transcriptome) are related to changes of respective messages in the translatome, total RNA and ribosome associated RNA from stress-treated and untreated cells were analyzed with yeast cDNA microarrays. To achieve this, fluorescently labeled (Cy5) cDNA was prepared from total RNA isolated from yeast cell extracts and from affinity-purified ribosomes, and each sample was mixed with differentially labeled (Cy3) cDNA prepared from a common reference RNA pool, and competitively hybridized on yeast cDNA microarrays. We performed five independent experiments with untreated cells grown in minimal medium (t=0 reference) and three or more independent biological replicates of treated cells. Altogether, we sampled the cell's response to five different "stress treatments" that were applied for relatively short periods of time (10 or 20 minutes) to avoid secondary effects triggered by transcriptional adaption. A stimulus or stress experiment design type is where that tests response of an organism(s) to stress/stimulus. e.g. osmotic stress, behavioral treatment Time: Duration of the treatment Growth Condition: Type of treatment stimulus_or_stress_design

2010-06-25 | E-GEOD-13653 | biostudies-arrayexpress

Affinity purification of ribosomes and associated RNAs using tagged Rpl16a and Rpl16b

Project description:In this study, we systematically identified RNAs associated with ribosomes. To identify ribosome associated RNAs, C-terminal ZZ-tagged Rpl16a or Rpl16b, expressed under control of thier native promoter, were affinity purified from whole cell extracts of cultures grown to mid-log phase in minimal medium. Extracts were incubated with immunoglobulin G (IgG) coupled microbeads, washed, and ribosomes were eluted by tobacco etch virus (TEV) protease treatment. We performed two biological replicates with each protein and analyzed the RNA content using oligo microarrays. Total RNA isolated from extracts of cells expressing Rpl16a-ZZ or Rpl16b-ZZ (input) and from the affinity-purified ribosomes was reverse transcribed with oligo(dT) primers. cDNA was labeled with Cy3 and Cy5 fluorescent dyes, respectively, and competitively hybridized to yeast oligo microarrays. Alternatively, RNA was isolated from sucrose gradient fractions containing 60S subunits, 80S monosomes and polysomes. Here, we performed four biological replicates. Analysis was done with oligo microarrays as described above. A strain or line experiment design type assays differences between multiple strains, cultivars, serovars, isolates, lines from organisms of a single species.

2009-05-12 | GSE13654 | GEO

Affinity purification of ribosomes and associated RNAs using tagged Rpl16a and Rpl16b

2010-06-25 | E-GEOD-13654 | biostudies-arrayexpress

RBP40_arrays

Project description:In this study, we systematically identified the RNAs associated with a selective sample of 40 of ~600 yeast RNA-binding proteins (RBPs). To identify RNAs associated with each putative RBP, C-terminal tandem affinity purification (TAP)-tagged proteins, expressed under control of their native promoters, were affinity purified from whole cell extracts of cultures grown to mid-log phase in rich medium [1-3]. Extracts were incubated with immunoglobulin G (IgG) agarose beads, washed, and ribonuclear protein complexes were eluted by tobacco etch virus (TEV) protease treatment (Text S2). We performed two to four independent isolations with each tagged strain. As controls, we performed 13 immunoaffinity purifications (IPs) of untagged strains to identify and exclude potential false-positive RNA targets. We purified total RNA from the whole-cell extracts and TEV-purified fractions, reverse transcribed with an amino-allyl-dUTP/dNTP mix, coupled the purified cDNA to Cy3 and Cy5 dyes, respectively, mixed the two differentially labeled cDNA pools, and then hybridized them to DNA microarrays (Datasets S1-S4). An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Genotype: strain expressing tagged version of RNA-BP Keywords: all_pairs

2008-10-28 | GSE13135 | GEO

RBP40_arrays

2010-06-25 | E-GEOD-13135 | biostudies-arrayexpress

Molecular profiling of reticular gigantocellularis neurons

Project description:we used technique that allows the molecular characterization of particular neuronal subpopulations based on their neuroanatomical projections and the locations of their cell bodies. This ‘retro-TRAP’ (translating ribosome affinity purification from retrogradely labeled neurons) approach relies on viral injection into an anatomical area targeted by the neurons of interest, followed by selective precipitation of ribosomes from retrogradely labeled cell bodies, and subsequent RNAseq analysis.

2018-11-01 | GSE115526 | GEO

Optimization of codon translation rates via tRNA modifications maintains proteome integrity

Project description:Proteins begin to fold as they emerge from translating ribosomes. The kinetics of ribosome transit along a given mRNA can influence nascent chain folding, but the extent to which individual codon translation rates impact proteome integrity remains unknown. Here, we show that slower decoding of discrete codons elicits widespread protein aggregation in vivo. Using ribosome profiling, we find that loss of anticodon wobble uridine (U34) modifications in a subset of tRNAs leads to ribosome pausing at their cognate codons in S. cerevisiae and C. elegans. Yeast cells lacking U34 modifications exhibit gene expression hallmarks of proteotoxic stress and accumulate aggregates of endogenous proteins with key cellular functions. Moreover, these cells are severely compromised in clearing stress-induced protein aggregates. Overexpression of hypomodified tRNAs alleviates ribosome pausing, concomitantly restoring protein homeostasis. Our findings demonstrate that modified U34 is an evolutionarily conserved accelerator of decoding and reveal an unanticipated role for tRNA anticodon modifications in maintaining proteome integrity. Ribosome profiling of wild-type and tRNA modification-deficient yeast and nematodes. Yeast samples were generated in various growth conditions (rich medium versus stress induced by treatment with diamide or rapamycin) and paired mRNA-Seq was performed on a subset of samples. Dataset contains three biological replicates for yeast samples and two biological replicates for nematode samples.

2015-06-04 | E-GEOD-67387 | biostudies-arrayexpress

Khd1p associated mRNAs

Project description:RNA-coimmunopurifications with TAP-tagged Khd1 protein from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control (Gerber et al., 2004). Cells were grown to midlog phase in rich media and harvested by centrifugation. TAP-tagged Khd1 was affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input) and from purified protein samples by phenol-chloroform extraction. 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 DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004. An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract. Strain Name: yeast strain with/without TAP-tagged Khd1 Keywords: all_pairs

2008-08-26 | GSE10279 | GEO

Affinity purification of ribosomes and associated RNAs from stress-treated cells using tagged Rpl16a and Rpl16b

Project description:This SuperSeries is composed of the following subset Series: GSE13653: Affinity purification of ribosomes and associated RNAs from stress-treated cells GSE13654: Affinity purification of ribosomes and associated RNAs using tagged Rpl16a and Rpl16b Refer to individual Series

2010-06-25 | E-GEOD-13682 | biostudies-arrayexpress

RNA-affinity isolations 13 RBPs

Project description:To identify RNAs specifically associated with potential RBPs, yeast cells expressing TAP-tagged RBP or the wild-type strain BY4741 (mock control) were grown to mid-log phase in rich medium and harvested by centrifugation. RNA affinity isolations were essentially performed as described (Gerber et al. 2004 PLoS Biol.; see protocol). In brief, TAP-tagged protein were captured from cell extracts with IgG coupled agarose beads (Sigma) and released by incubation with a site-specific protease (AcTEV, Sigma). from the extract (input) and from the affinity isolates was purified with the RNeasy Mini/ Micro 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. Antigenic peptide used in IP: TAP-tag An all pairs experiment design type is where all labeled extracts are compared to every other labeled extract.

2010-12-06 | E-GEOD-21864 | biostudies-arrayexpress

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