Affinity purification of ribosomes to access the translatome

Halbeisen, Regula E.; Scherrer, Tanja; Gerber, André P.

doi:10.1016/j.ymeth.2009.04.003

Cited by 46 publications

(46 citation statements)

References 28 publications

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“…Arrays were processed and hybridized with fluorescently labeled cDNAs as described previously [47]. For RIP-Chip experiments, 5 mg of total RNA isolated from the extract (input) and up to 50% (,500 ng) of the affinity purified RNA were reverse transcribed in the presence of 5-(3-aminoallyl)-dUTP and natural dNTPs with a mixture of randome nonamer and dT(20)V primers, and cDNAs were covalently linked to Cy3 and Cy5 NHS-monoesters (GE HealthSciences Cat# RPN5661), respectively, and competitively hybridized on yeast oligo arrays at 42uC for 14 hours in formamide-based hybridization buffer.…”

Section: Dna Microarrays and Data Analysismentioning

confidence: 99%

Environmental Regulation of Prions in Yeast

2014

Investigations in Yeast Functional Genomics and Molecular Biology

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Hundreds of RNA-binding proteins (RBPs) control diverse aspects of post-transcriptional gene regulation. To identify novel and unconventional RBPs, we probed high-density protein microarrays with fluorescently labeled RNA and selected 200 proteins that reproducibly interacted with different types of RNA from budding yeast Saccharomyces cerevisiae. Surprisingly, more than half of these proteins represent previously known enzymes, many of them acting in metabolism, providing opportunities to directly connect intermediary metabolism with posttranscriptional gene regulation. We mapped the RNA targets for 13 proteins identified in this screen and found that they were associated with distinct groups of mRNAs, some of them coding for functionally related proteins. We also found that overexpression of the enzyme Map1 negatively affects the expression of experimentally defined mRNA targets. Our results suggest that many proteins may associate with mRNAs and possibly control their fates, providing dense connections between different layers of cellular regulation.

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Section: Dna Microarrays and Data Analysismentioning

confidence: 99%

Environmental Regulation of Prions in Yeast

2014

Investigations in Yeast Functional Genomics and Molecular Biology

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“…3B). In one application, the ribosomal protein Rpl16 in yeast was modified by addition of a protein A tag to allow purification of mRNAs associated with ribosomes (Halbeisen et al 2009). Similarly, a hemagglutinin (HA) tag was used to mark ribosomal protein Rpl22 in mouse (Sanz et al 2009).…”

Section: Genome-wide Analysis Of Translational Activitymentioning

confidence: 99%

Toward a Genome-Wide Landscape of Translational Control

Larsson

Tian

Sonenberg

2012

Cold Spring Harbor Perspectives in Biology

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“…GSE27312. Procession of yeast oligo microarrays and hybridization with fluorescently labeled cDNAs was essentially performed as described (Halbeisen et al 2009). Five micrograms of total RNA isolated from the extract (input) and 50% (z500 ng) of the affinity purified RNA was reverse transcribed with a mixture of random nonamer and dT20V primers in the presence of 5-(3-aminoallyl)-dUTP and natural dNTPs; cDNAs were covalently linked to Cy3 and Cy5 NHS-monoesters (GE HealthSciences, catalog no.…”

Section: Dna Microarrays and Data Analysismentioning

confidence: 99%

La-motif–dependent mRNA association with Slf1 promotes copper detoxification in yeast

Schenk

Meinel²,

Sträßer³

et al. 2012

RNA

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The La-motif (LAM) is an ancient and ubiquitous RNA-binding domain defining a superfamily of proteins, which comprises the genuine La proteins and La-related proteins (LARPs). In contrast to La, which binds and stabilizes pre-tRNAs and other RNA polymerase III transcripts, data on function and RNA targets of the LARPs have remained scarce. We have undertaken a global approach to elucidate the previously suggested role of the yeast LARP Slf1p in copper homeostasis. By applying RNA-binding protein immunopurification-microarray (RIP-Chip) analysis, we show that Slf1p and its paralog Sro9p copurify with overlapping sets of hundreds of functionally related mRNAs, including many transcripts coding for ribosomal proteins and histones. Interestingly, among these potential RNA targets were also mRNAs coding for proteins critical for protection of cells against elevated copper concentrations. Mutations introduced in the conserved aromatic patch of the LAM in Slf1p drastically impaired both association with its targets and Slf1-mediated protection of cells against toxic copper concentrations. Furthermore, we show that Slf1p stabilizes copper-related mRNA targets in a LAM-dependent manner. These results provide the first evidence for post-transcriptional regulation of factors/pathways implicated in copper homeostasis by a cytoplasmic RBP.

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Affinity purification of ribosomes to access the translatome

Cited by 46 publications

References 28 publications

Environmental Regulation of Prions in Yeast

Environmental Regulation of Prions in Yeast

Toward a Genome-Wide Landscape of Translational Control

La-motif–dependent mRNA association with Slf1 promotes copper detoxification in yeast

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