Genome-wide identification of targets of the drosha–pasha/DGCR8 complex

Abstract

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 cotranscriptionally. 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 kb as revealed by both the Drosha knockdown 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 59 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.

Viability, Longevity, and Egg Production of Drosophila melanogaster Are Regulated by the miR-282 microRNA

“…The 39-RACE technique using primers specific for the adapter sequence of the GeneRacer oligo(dT) primer revealed that the poly(A) tail at the 39 end is in the 3L: 3,254,504 genomic position, which is 3446 bp downstream from the last nucleotide of the mature miR-282. These results suggest that the mir-282 primary transcript has a 59 cap and a 39-poly(A) sequence and that it is 4918 bp long, which corresponds to the size predicted previously (Kadener et al 2009) (Figure 1A).…”

“…We demonstrate that the predicted mir-282 gene is actively transcribed and that the lack of miR-282 leads to pupal semi-lethality and reduced egg production while ubiquitous, ectopic overexpression of miR-282 causes lethality at the larval stage. Our results, together with a finding that the knockdown of Drosha results in the accumulation of mir-282 pri-miRNA (KADENER et al 2009), provide molecular evidence for the existence of the predicted mir-282 gene in Drosophila melanogaster.…”

“…Although it is still possible that the mir-282 transcript is a splice form of a distant gene, the well-defined 59 end of mir-282 and the class I insulator-defined regulatory boundary predicted upstream from mir-282 by the modENCODE database (Négre et al 2010), as well as a recent report on mir-282 being the target of the Drosha-Pasha complex (instead of the splicing machinery) (Kadener et al 2009), all argue for an independent mir-282 transcription unit.…”

Identification of microprocessor-dependent cancer cells allows screening for growth-sustaining micro-RNAs

“…It should also be noted that the microprocessor is able to target and cleave the DGCR8 mRNA (Han et al, 2009;Kadener et al, 2009;Triboulet et al, 2009). Although targeting of other mRNAs by the microprocessor (Kadener et al, 2009) is still controversial (Shenoy and Blelloch, 2009), we cannot exclude that inhibiting Drosha or DGCR8 could modify the expression of non-pri-miRNA RNAs.…”

“…Although targeting of other mRNAs by the microprocessor (Kadener et al, 2009) is still controversial (Shenoy and Blelloch, 2009), we cannot exclude that inhibiting Drosha or DGCR8 could modify the expression of non-pri-miRNA RNAs.…”