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Unknown,Transcriptomics,Genomics,Proteomics

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RNAi of human cells after Drosha knockdown reduces viability but does not affect snRNA or rRNA synthesis or processing

ABSTRACT: We asked whether the human drosha protein, an RNase III homolog known to process microRNAs (miRNAs), might also be a small nuclear RNA (snRNA) 3' processing factor. Using retroviral siRNA silencing constructs, we stably knocked down drosha protein to nearly undetectable levels. Knockdown cells exhibited reduced growth rates and viability compared to controls, but no accumulation of unprocessed U2 snRNA precursors. In fungi, RNase III homologs process rRNA precursors and certain mRNAs. Although rRNA processing appears to be normal in the drosha knockdown cells, expression microarray analysis revealed misregulation of several mRNAs involved in cell growth and proliferation. Curiously, drosha knockdown appeared to downregulate the predicted mRNA targets of several miRNAs Experiment Overall Design: The experimental goal was to evaluate gene expression changes induced by siRNA knockdown of drosha. Four samples of HeLa cells were transfected with retroviral siRNA expression vectors: two replicates of an anti-GFP siRNA vector (siGFP) and two different anti-drosha siRNA vectors (sidroshaB and sidroshaC). Cells were selected with puromycin 24 hours after transfection and harvested 72 hours after transfection. Trizol-harvested RNA was processed with standard Affymetrix protocols and hybridized to U133Plus2.0 GeneChips. Signals were scaled to an arbitrary global mean value of 800.

ORGANISM(S): Homo sapiens

SUBMITTER: John Newman

PROVIDER: E-GEOD-6767 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Drosha knockdown in human cells reduces viability but does not affect snRNA or rRNA synthesis or processing

Project description:We asked whether the human drosha protein, an RNase III homolog known to process microRNAs (miRNAs), might also be a small nuclear RNA (snRNA) 3' processing factor. Using retroviral siRNA silencing constructs, we stably knocked down drosha protein to nearly undetectable levels. Knockdown cells exhibited reduced growth rates and viability compared to controls, but no accumulation of unprocessed U2 snRNA precursors. In fungi, RNase III homologs process rRNA precursors and certain mRNAs. Although rRNA processing appears to be normal in the drosha knockdown cells, expression microarray analysis revealed misregulation of several mRNAs involved in cell growth and proliferation. Curiously, drosha knockdown appeared to downregulate the predicted mRNA targets of several miRNAs Keywords: siRNA knockdown

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