Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNAi profiling of Drosophila Schneider S2 cells treated with dsRNA vs. GFP

ABSTRACT: Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals that endogenous small interfering RNAs (siRNAs) are produced via bidirectional transcription. >100 cis-NATs with overlapping 3' exons generate 21-nt, Dicer-2 (Dcr-2)Âdependent, 3'-end modified siRNAs. To determine whether any co-expressed cisNATs are denied entry into the RNAi pathway, we analyzed the gene expression profile of S2 cells. The analysis suggested that the processing of cis-NATs by RNAi are actively restricted, and the selected loci are enriched for nucleic acidÂbased functions and include Argonaute-2 (AGO2) itself. Experiment Overall Design: Drosophila Schneider cells (S2) were treated with dsRNA against GFP for 8 days. The treatment was done in duplicate. Total RNA was extracted from the dsRNA treated cells using trizol. Experiment Overall Design: Gene expression analysis was conducted using Drosophila Genome 2.0 GenechipÂ® arrays (Affymetrix, Santa Clara, CA). Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3Â¹ Amplification One-Cycle Target labeling kit according to manufacturerÂ¹s protocol. For each array, 10ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000 and data was obtained using the GenechipÂ® Operating Software (Version 1.2.0.037).

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Sudha Balla

PROVIDER: E-GEOD-11370 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster.

Okamura Katsutomo K Balla Sudha S Martin Raquel R Liu Na N Lai Eric C EC

Nature structural & molecular biology 20080525 6

Cis-natural antisense transcripts (cis-NATs) have been speculated to be substrates for endogenous RNA interference (RNAi), but little experimental evidence for such a pathway in animals has been reported. Analysis of massive Drosophila melanogaster small RNA data sets now reveals two mechanisms that yield endogenous small interfering RNAs (siRNAs) via bidirectional transcription. First, >100 cis-NATs with overlapping 3' exons generate 21-nt, and, based on previously published small RNA data [cor ...[more]

PMID: 18500351

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Project description:Metazoan transcription is controlled through either coordinated recruitment of transcription machinery to the gene promoter, or subsequently, through regulated pausing of RNA polymerase II (Pol II) in early elongation. We report that a key difference between genes that use these distinct regulatory strategies lies in the chromatin architecture specified by their DNA sequences. Pol II pausing is prominent at highly-regulated genes whose sequences inherently disfavor nucleosome formation within the gene, but favor nucleosomal occlusion of the promoter. Pausing of polymerase maintains these genes in an active state by inhibiting the formation of repressive promoter chromatin. In contrast, promoters of housekeeping genes that lack paused Pol II are deprived of nucleosomes regardless of polymerase binding, but show higher nucleosome occupancy downstream. Our results suggest that the â??defaultâ?? chromatin state of a gene instructs its regulation, and that highly-regulated promoters have evolved to encourage competition between nucleosomes and paused Pol II for promoter occupancy. Drosophila melanogaster S2 cells (Drosophila Genomics Resource Center) were untreated, or treated with dsRNA for 96 hours, as described in Armknecht, S. et al. (2005) Methods in Enzymology, 392: pp. 55-73. Total RNA was then extracted using the RNeasy RNA extraction kit, with on column DNAse digestion (Qiagen), according to manufacturerâ??s protocol. Gene expression analysis was conducted using Drosophila Genome 2.0 GenechipÂ® arrays (Affymetrix, Santa Clara, CA). Starting with 1ug of total RNA, biotin-labeled cRNA was produced using the Affymetrix 3â?? Amplification One-Cycle Target labeling kit according to manufacturerâ??s protocol. For each array, 10ug of amplified cRNAs were fragmented and hybridized to the array for 16 hours in a rotating hybridization oven using the Affymetrix Eukaryotic Target Hybridization Controls and protocol. Slides were stained and washed as indicated in the Antibody Amplification Stain for Eukaryotic Targets protocol using the Affymetrix Fluidics Station FS450. Arrays were then scanned with an Affymetrix Scanner 3000 and data was obtained using either GenechipÂ® Operating Software (Version 1.2.0.037) or Affymetrix GeneChip Command Console (V. 1.1) and imported into the Rosetta Resolver system (Version 6.0) as outlined in the scan and data processing protocols.

Dataset Information

RNAi profiling of Drosophila Schneider S2 cells treated with dsRNA vs. GFP

Publications

Two distinct mechanisms generate endogenous siRNAs from bidirectional transcription in Drosophila melanogaster.

Similar Datasets

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