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PLoS One. 2013 Sep 3;8(9):e74296. doi: 10.1371/journal.pone.0074296. eCollection 2013.

AutomiG, a biosensor to detect alterations in miRNA biogenesis and in small RNA silencing guided by perfect target complementarity.

Author information

1
Drosophila Genetics and Epigenetics, Laboratory of Developmental Biology, CNRS UMR7622, Université Pierre et Marie Curie, Paris, France.

Abstract

Defects in miRNA biogenesis or activity are associated to development abnormalities and diseases. In Drosophila, miRNAs are predominantly loaded in Argonaute-1, which they guide for silencing of target RNAs. The miRNA pathway overlaps the RNAi pathway in this organism, as miRNAs may also associate with Argonaute-2, the mediator of RNAi. We set up a gene construct in which a single inducible promoter directs the expression of the GFP protein as well as two miRNAs perfectly matching the GFP sequences. We show that self-silencing of the resulting automiG gene requires Drosha, Pasha, Dicer-1, Dicer-2 and Argonaute-2 loaded with the anti-GFP miRNAs. In contrast, self-silencing of the automiG gene does not involve Argonaute-1. Thus, automiG reports in vivo for both miRNA biogenesis and Ago-2 mediated silencing, providing a powerful biosensor to identify situations where miRNA or siRNA pathways are impaired. As a proof of concept, we used automiG as a biosensor to screen a chemical library and identified 29 molecules that strongly inhibit miRNA silencing, out of which 5 also inhibit RNAi triggered by long double-stranded RNA. Finally, the automiG sensor is also self-silenced by the anti-GFP miRNAs in HeLa cells and might be easily used to identify factors involved in miRNA biogenesis and silencing guided by perfect target complementarity in mammals.

PMID:
24019960
PMCID:
PMC3760873
DOI:
10.1371/journal.pone.0074296
[Indexed for MEDLINE]
Free PMC Article

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