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Co-immunoprecipitation of RNAs with RNA helicase A (RHA)

ABSTRACT: Given that RHA regulates translation by binding a PCE located at the 5’ UTR of the target transcripts a genome-wide screen was performed to identify mRNAs that bind RHA in vivo. The results from duplicate experiments with samples obtained in two independent RNA immunoprecipitations identified 407 transcripts that co-immunoprecipitate with FLAG RHA. Keywords: gene expression array-based

ORGANISM(S): Chlorocebus aethiops Macaca mulatta

PROVIDER: GSE14056 | GEO | 2008/12/20

SECONDARY ACCESSION(S): PRJNA114525

REPOSITORIES: GEO

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Co-immunoprecipitation of RNAs with RNA helicase A (RHA)

Project description:Given that RHA regulates translation by binding a PCE located at the 5’ UTR of the target transcripts a genome-wide screen was performed to identify mRNAs that bind RHA in vivo. The results from duplicate experiments with samples obtained in two independent RNA immunoprecipitations identified 407 transcripts that co-immunoprecipitate with FLAG RHA. Keywords: gene expression array-based Since N-terminal FLAG tagged RHA specifically co-immunoprecipitates PCE-containing mRNAs COS cells were transfected with a CMV-FLAG-RHA construct. Cytoplasmic lysates were pooled and immunoprecipitated with anti-FLAG beads. RNA was extracted from the immunoprecipitate and used to probe rhesus macaque Affimetrix expression arrays. Steady state mRNA levels were monitored on a separate array probed with total RNA.

2008-12-19 | E-GEOD-14056 | biostudies-arrayexpress

RNA immunoprecipitation to identify RHA-binding transcripts in HEK293 cells

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RNA immunoprecipitation to identify RHA-binding transcripts in HEK293 cells

Project description:Given that RHA regulates translation by binding a PCE located at the 5’ UTR of the target transcripts a genome-wide screen was performed to identify mRNAs that bind RHA in vivo. The results from four experiments with samples obtained in four independent RNA immunoprecipitations identified 375 transcripts that co-immunoprecipitate with FLAG RHA. Since N-terminal FLAG tagged RHA specifically co-immunoprecipitates PCE-containing mRNAs HEK293 cells were transfected with a CMV-FLAG-RHA construct. Cytoplasmic lysates were immunoprecipitated with anti-FLAG beads. RNA was extracted from the immunoprecipitate and used to probe a human Agilent expression arrays. An immunoprecipitation with cells transfected with empty FLAG plasmid was used as negative control.

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Changes in polysome loading as a consequence of RHA downregulation

Project description:RNA helicase A (RHA) binds its target transcripts at the post-transcriptional control element (PCE) located in the 5’ untranslated region (UTR). This interaction represents an “RNA switch” that regulates protein synthesis. Down regulation of RHA by siRNAs was used to identify transcripts with RHA-dependent translation. Reduced accumulation of RNA in polysomes was monitored with microarrays. Changes in cytoplasmic RNA steady state abundance was monitored as well. Sixty nine genes exhibit decreased transcript polysome association when subjected to RHA downregulation. A majority of the transcripts that experienced a reduction in the polysome fraction had no significant change in their cytoplasmic abundance (45 genes). Keywords: gene expression array-based

2008-12-20 | GSE14055 | GEO

Changes in polysome loading as a consequence of RHA downregulation [Agilent]

Project description:RNA helicase A (RHA) binds its target transcripts at the post-transcriptional control element (PCE) located in the 5’ untranslated region (UTR). This interaction represents an “RNA switch” that regulates protein synthesis. Down regulation of RHA by siRNAs was used to identify transcripts with RHA-dependent translation. Reduced accumulation of RNA in polysomes was monitored with microarrays. Cytoplasmic lysates of cells treated with RHA targeted or non-silencing control siRNAs were separated by sucrose density gradient centrifugation. Ribosomal RNA profiles were generated, containing heavy polysomes were collected, and RNA was extracted.

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Changes in polysome loading as a consequence of RHA downregulation

Project description:RNA helicase A (RHA) binds its target transcripts at the post-transcriptional control element (PCE) located in the 5â?? untranslated region (UTR). This interaction represents an â??RNA switchâ?? that regulates protein synthesis. Down regulation of RHA by siRNAs was used to identify transcripts with RHA-dependent translation. Reduced accumulation of RNA in polysomes was monitored with microarrays. Changes in cytoplasmic RNA steady state abundance was monitored as well. Sixty nine genes exhibit decreased transcript polysome association when subjected to RHA downregulation. A majority of the transcripts that experienced a reduction in the polysome fraction had no significant change in their cytoplasmic abundance (45 genes). Keywords: gene expression array-based Cytoplasmic lysates of cells treated with RHA targeted or non-silencing control siRNAs were separated by sucrose density gradient centrifugation. Ribosomal RNA profiles were generated, fractions containing polysomes were collected, and RNA was extracted.

2010-05-15 | E-GEOD-14055 | biostudies-arrayexpress

Identification of transcripts regulated by RNA helicase A (RHA)

Project description:This SuperSeries is composed of the following subset Series: GSE14055: Changes in polysome loading as a consequence of RHA downregulation GSE14056: Co-immunoprecipitation of RNAs with RNA helicase A (RHA) Refer to individual Series

2008-12-19 | E-GEOD-14057 | biostudies-arrayexpress

Identification of RNA helicase A translation target mRNAs at the intersection of RNA precipitation and polysome arrays

Project description:This SuperSeries is composed of the following subset Series: GSE23669: Changes in polysome loading as a consequence of RHA downregulation [Agilent] GSE23688: RNA immunoprecipitation to identify RHA-binding transcripts in HEK293 cells Refer to individual Series

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