Identification of RNA helicase A translation target mRNAs at the intersection of RNA precipitation and polysome arrays
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Kathleen Boris-Lawrie
PROVIDER: E-GEOD-23689 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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.
2010-08-19 | E-GEOD-23669 | biostudies-arrayexpress
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.
2010-08-19 | E-GEOD-23688 | biostudies-arrayexpress
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
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
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.
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Project description:Protein translation factors play crucial roles in a variety of stress responses. Here, we show that the eukaryotic elongation factor 1Bdelta (eEF1Bdelta) changes its structure and function from a translation factor into a heat shock response transcription factor by alternative splicing. While eEF1Bdelta is specifically localized in the cytoplasm, the long isoform of eEF1Bdelta (eEF1BdeltaL) is localized in the nucleus and induces heat shock element (HSE)-containing genes in cooperation with heat-shock transcription factor 1 (HSF1). Moreover, the N-terminal domain of eEF1BdeltaL binds with NF-E2-related factor 2 (Nrf2) and induces stress response heme oxygenase 1 (HO-1). Specific inhibition of eEF1BdeltaL with siRNA completely inhibits Nrf2-dependent HO-1 induction. In addition, eEF1BdeltaL directly binds to HSE oligo DNA in vitro and associates with HSE containing the HO-1-enhancer region in vivo. Thus, the transcriptional role of eEF1BdeltaL could provide new insights into the molecular mechanism of stress responses. We performed microarray analysis to compare the gene expression induced by eEF1Bdelta1 or eEF1BdeltaL overexpression. HEK293 cells transfected with expression plasmids encoding flag-tagged-eEF1Bdelta1 or eEF1BdeltaL protein
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Project description:Isolation of IMP1 bound mRNAs. Flag-tagged IMP1 was expressed in HEK293 cells. Flag tagged IMP1 was immunoprecipitated and mRNAs isolated. As controls HEK293 cells that do not express Flag-tagged IMP1 was included.
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