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| Status |
Public on Dec 25, 2008 |
| Title |
Genome wide identification of targets of the drosha-pasha/DGCR8 complex |
| Organism |
Drosophila melanogaster |
| Experiment type |
Expression profiling by genome tiling array
Genome binding/occupancy profiling by genome tiling array
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| Summary |
Drosha is a type III RNAse, which plays a critical role in miRNA biogenesis. Drosha and its double-stranded RNA-binding partner protein Pasha/DGCR8 likely recognize and cleave miRNA precursor RNAs or pri-miRNA hairpins co-transcriptionally. To identify RNAs processed by Drosha, we used tiling microarrays to examine transcripts after depletion of drosha mRNA with dsRNA in Drosophila Schneider S2 cells. This strategy identified 137 Drosha-regulated RNAs, including 11 putative pri-miRNAs comprising 15 annotated miRNAs. Most of the identified pri-miRNAs seem extremely large, >10 kilobases as revealed by both the Drosha knock down strategy and by RNA PolII chromatin IP followed by Drosophila tiling microarrays. Surprisingly, more than a hundred additional RNAs not annotated as miRNAs are under Drosha control and are likely to be direct targets of Drosha action. This is because many of them encode annotated genes, and unlike bona fide pri-miRNAs, they are not affected by depletion of the miRNA processing factor, dicer-1. Moreover, application of the evofold analysis software indicates that at least 25 of the Drosha-regulated RNAs contain evolutionarily conserved hairpins similar to those recognized by the Drosha-Pasha/DGCR8 complex in pri-miRNAs. One of these hairpins is located in the 5′ UTR of both pasha and mammalian DGCR8. These observations suggest that a negative feedback loop acting on pasha mRNA may regulate the miRNA-biogenesis pathway: i.e., excess Drosha cleaves pasha/DGCR8 primary transcripts and leads to a reduction in pasha/DGCR8 mRNA levels and Pasha/DGCR8 synthesis.
Keywords: time course, ChIP-chip
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| Overall design |
S2 cells were depleted of drosha or dicer mRNA with dsRNA. Total RNA was then converted to cDNA and hybridized to Affymetrix tiling arrays to identify pri-miRNAs and drosha regulated RNAs. RNA PolII chromatin IP genomic DNA was also hybridized to Affymetix tiling arrays to support pri-miRNA targets from drosha knockdowns. Two replicates were used for each sample.
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| Contributor(s) |
Kadener S, Rodriguez J, Abruzzi K, Khodor YL, Sugino K, Marr M, Nelson S, Rosbash M |
| Citation(s) |
19223442 |
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| Submission date |
Dec 24, 2008 |
| Last update date |
Jul 08, 2015 |
| Contact name |
Joseph Rodriguez |
| Organization name |
Brandeis University
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| Department |
Biology
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| Lab |
Rosbash
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| Street address |
415 South Street
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| City |
Waltham |
| ZIP/Postal code |
02453 |
| Country |
USA |
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| Platforms (2) |
| GPL5919 |
[Dm35b_MR] Affymetrix Drosophila Tiling 1.0R Array |
| GPL6629 |
[DM_tiling2_MR] Affymetrix Drosophila Tiling 2.0R Array |
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| Samples (7)
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| GSM356322 |
S2 control,biological replicate 1 |
| GSM356323 |
S2 control,biological replicate 2 |
| GSM356324 |
S2 dicer knockdown,biological replicate 1 |
| GSM356325 |
S2 dicer knockdown,biological replicate 2 |
| GSM356326 |
S2 drosha knockdown,biological replicate 1 |
| GSM356327 |
S2 drosha knockdown,biological replicate 2 |
| GSM356328 |
S2 Pol II IP vs S2 cells, ChIP input |
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| Relations |
| BioProject |
PRJNA111341 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE14215_RAW.tar |
373.2 Mb |
(http)(custom) |
TAR (of BAR, CEL) |
| Processed data provided as supplementary file |
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