Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

RNAi knock down of dU2AF50 in Drosophila mel2 cells and profiling of nuclear and cytoplasmic fractions

ABSTRACT: Nuclear and cytoplasmic RNA were extracted by hypotonic cell lysis from D.-mel2 cell knock down by RNAi against dU2AF50 or with non-specific dsRNA.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Marco Blanchette

PROVIDER: E-GEOD-1344 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Genome-wide analysis reveals an unexpected function for the Drosophila splicing factor U2AF50 in the nuclear export of intronless mRNAs.

Blanchette Marco M Labourier Emmanuel E Green Richard E RE Brenner Steven E SE Rio Donald C DC

Molecular cell 20040601 6

The protein factor U2AF is an essential component required for pre-mRNA splicing. Mutations identified in the S. pombe large U2AF subunit were used to engineer transgenic Drosophila carrying temperature-sensitive U2AF large subunit alleles. Mutant recombinant U2AF heterodimers showed reduced polypyrimidine tract RNA binding at elevated temperatures. Genome-wide RNA profiling comparing wild-type and mutant strains identified more than 400 genes differentially expressed in the dU2AF50 mutant flies ...[more]

PMID: 15200955

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Project description:To accelerate previous RNA structure probing approaches, which focus on analyzing one RNA sequence at a time, we have developed FragSeq, a high-throughput RNA structure probing method that uses high-throughput RNA sequencing to identify single-stranded RNA (ssRNA) regions from fragments generated by nuclease P1, which is specific for single-stranded nucleic acids. In the accompanying study, we show that we can accurately and simultaneously map ssRNA regions in multiple non-coding RNAs with known structure in experiments probing the entire mouse nuclear transcriptome. We carried out probing in two cell types to assess reproducibility. We also identified and experimentally validated structured regions in ncRNAs never previously probed. We examined mouse nuclear RNA from two cell types: undifferentiated embryonic stem cells (UNDIFF) and cells differentiated into neural precursors (D5NP). For each cell type, nuclear RNA was purified and deproteinized, denatured, and refolded in vitro, from which we prepared three barcoded samples: "nuclease" (RNA partially digested with P1 ssRNA-specific nuclease, yielding 5'-PO4/3'-OH end chemistry at each cleavage site), "control" (control for "nuclease" sample to idenfity endogenous 5'-PO4/3'-OH), and "PNK" (same as "control" but followed by a polynucleotide kinase treatment to convert 5'-OH/3'-cyclic-phosphate ends to clonable 5'-PO4/3'-OH ends). Resulting RNA fragments were cloned using the SOLiD Small RNA Expression Kit (SREK) protocol, which ligates linkers only to 5'-PO4/3'-OH containing RNA, enriching for clones of products resulting from P1 cleavage in "nuclease" sample and selecting against random degradation. Two cell types, three treatments each, thus resulted in six barcoded samples total (barcodes 01, 02, 04, 05, 07, 08). Four other barcoded samples were prepared for separate experiments not used in our study (barcodes 03, 06, 09, 10), so their preparation is not described here. The total run of ten barcodes was done on the ABI SOLiD3 platform and a custom algorithm (FragSeq v0.0.1) was used to compute "cutting scores" (as described in our paper) that show ssRNA regions in hundreds of ncRNAs.

Dataset Information

RNAi knock down of dU2AF50 in Drosophila mel2 cells and profiling of nuclear and cytoplasmic fractions

Publications

Genome-wide analysis reveals an unexpected function for the Drosophila splicing factor U2AF50 in the nuclear export of intronless mRNAs.

Similar Datasets

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