Transcriptomics

Dataset Information

2

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


ABSTRACT: 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. strain_or_line_design

ORGANISM(S): Saccharomyces cerevisiae  

SUBMITTER: Regula Halbeisen   Andre Gerber 

PROVIDER: E-GEOD-13654 | ArrayExpress | 2010-06-25

SECONDARY ACCESSION(S): GSE13654PRJNA113917

REPOSITORIES: GEO, ArrayExpress

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Publications

Stress-dependent coordination of transcriptome and translatome in yeast.

Halbeisen Regula E RE   Gerber AndrĂ© P AP  

PLoS biology 20090505 5


Cells rapidly alter gene expression in response to environmental stimuli such as nutrients, hormones, and drugs. During the imposed "remodeling" of gene expression, changes in the levels of particular mRNAs do not necessarily correlate with those of the encoded proteins, which could in part rely on the differential recruitment of mRNAs to translating ribosomes. To systematically address this issue, we have established an approach to rapidly access the translational status of each mRNA in the yea  ...[more]

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