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

Carré C, Jacquier C, Bougé AL, de Chaumont F, Besnard-Guerin C, Thomassin H, Pidoux J, Da Silva B, Chalatsi E, Zahra S, Olivo-Marin JC, Munier-Lehmann H, Antoniewski C - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

Show MeSH

Related in: MedlinePlus

The CMV-automiG construct is a sensor of the human miRNA pathway.(A) HeLa cells transiently co-transfected with CMV-automiG and the indicated siRNAs were grown for 72 h and GFP expression was analyzed by western blot. The γ-Tubulin protein was used as a loading control. (B) Suppression of the CMV-automiG silencing by the #2516-4080 and #3601-0038 compounds. HeLa cells transiently transfected with CMV-automiG for 24 h were soaked for 48 additional hours with the indicated compounds or DMSO alone as a control, and GFP expression was analyzed by western blot. The γ-Tubulin protein was used as a loading control. The effects of the compounds #D010-0185 and #D094-0021 on GFP expression could not be assayed due to their toxicity.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3760873&req=5

pone-0074296-g008: The CMV-automiG construct is a sensor of the human miRNA pathway.(A) HeLa cells transiently co-transfected with CMV-automiG and the indicated siRNAs were grown for 72 h and GFP expression was analyzed by western blot. The γ-Tubulin protein was used as a loading control. (B) Suppression of the CMV-automiG silencing by the #2516-4080 and #3601-0038 compounds. HeLa cells transiently transfected with CMV-automiG for 24 h were soaked for 48 additional hours with the indicated compounds or DMSO alone as a control, and GFP expression was analyzed by western blot. The γ-Tubulin protein was used as a loading control. The effects of the compounds #D010-0185 and #D094-0021 on GFP expression could not be assayed due to their toxicity.

Mentions: As the mechanisms of both miRNA biogenesis and intron splicing appear to be conserved throughout evolution, the automiG construct could also provide a sensor system for miRNA biogenesis and activity in mammals. To test this possibility, we generated a CMV-automiG variant construct under the control of the CMV promoter which drives transcription in mammalian cell. When transfected alone or cotransfected with a scramble siRNA control in HeLa cells, the CMV-automiG construct expressed barely detectable levels of GFP protein (Fig. 8A). In striking contrast, cotransfections with siRNAs targeting the human key components of miRNA biogenesis, Drosha and its cofactor DGCR8, were associated with significant GFP induction (Fig. 8A), strongly suggesting that the CMV-automiG construct is indeed a biosensor for miRNA biogenesis in mammals. Finally, we selected the compounds #2516-4080 and #3601-0038, which inhibit miRNA silencing but not RNAi in S2R+ Drosophila cells, and the compounds #D010-0185 and #D094-0021, which inhibit both silencing pathways (Fig. 6 and 7), and we tested their effects on the CMV-automiG in HeLa cells. Twenty-four hours after transfection of HeLa cells with the CMV-automiG construct, the compounds were added to the medium for 48 h. The effect of the compounds #D010-0185 and #D094-0021 on GFP expression could not be assayed due to their toxicity (data not shown). In contrast, the compounds #2516-4080 and #3601-0038 were associated with significant GFP induction as compared to the DMSO control (Fig. 8B). The finding that compounds desilencing the automiG construct in Drosophila cells also desilence the CMV-automiG variant in HeLa cells further validates automiG as a versatile biosensor for miRNA biogenesis and activity.


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

Carré C, Jacquier C, Bougé AL, de Chaumont F, Besnard-Guerin C, Thomassin H, Pidoux J, Da Silva B, Chalatsi E, Zahra S, Olivo-Marin JC, Munier-Lehmann H, Antoniewski C - PLoS ONE (2013)

The CMV-automiG construct is a sensor of the human miRNA pathway.(A) HeLa cells transiently co-transfected with CMV-automiG and the indicated siRNAs were grown for 72 h and GFP expression was analyzed by western blot. The γ-Tubulin protein was used as a loading control. (B) Suppression of the CMV-automiG silencing by the #2516-4080 and #3601-0038 compounds. HeLa cells transiently transfected with CMV-automiG for 24 h were soaked for 48 additional hours with the indicated compounds or DMSO alone as a control, and GFP expression was analyzed by western blot. The γ-Tubulin protein was used as a loading control. The effects of the compounds #D010-0185 and #D094-0021 on GFP expression could not be assayed due to their toxicity.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3760873&req=5

pone-0074296-g008: The CMV-automiG construct is a sensor of the human miRNA pathway.(A) HeLa cells transiently co-transfected with CMV-automiG and the indicated siRNAs were grown for 72 h and GFP expression was analyzed by western blot. The γ-Tubulin protein was used as a loading control. (B) Suppression of the CMV-automiG silencing by the #2516-4080 and #3601-0038 compounds. HeLa cells transiently transfected with CMV-automiG for 24 h were soaked for 48 additional hours with the indicated compounds or DMSO alone as a control, and GFP expression was analyzed by western blot. The γ-Tubulin protein was used as a loading control. The effects of the compounds #D010-0185 and #D094-0021 on GFP expression could not be assayed due to their toxicity.
Mentions: As the mechanisms of both miRNA biogenesis and intron splicing appear to be conserved throughout evolution, the automiG construct could also provide a sensor system for miRNA biogenesis and activity in mammals. To test this possibility, we generated a CMV-automiG variant construct under the control of the CMV promoter which drives transcription in mammalian cell. When transfected alone or cotransfected with a scramble siRNA control in HeLa cells, the CMV-automiG construct expressed barely detectable levels of GFP protein (Fig. 8A). In striking contrast, cotransfections with siRNAs targeting the human key components of miRNA biogenesis, Drosha and its cofactor DGCR8, were associated with significant GFP induction (Fig. 8A), strongly suggesting that the CMV-automiG construct is indeed a biosensor for miRNA biogenesis in mammals. Finally, we selected the compounds #2516-4080 and #3601-0038, which inhibit miRNA silencing but not RNAi in S2R+ Drosophila cells, and the compounds #D010-0185 and #D094-0021, which inhibit both silencing pathways (Fig. 6 and 7), and we tested their effects on the CMV-automiG in HeLa cells. Twenty-four hours after transfection of HeLa cells with the CMV-automiG construct, the compounds were added to the medium for 48 h. The effect of the compounds #D010-0185 and #D094-0021 on GFP expression could not be assayed due to their toxicity (data not shown). In contrast, the compounds #2516-4080 and #3601-0038 were associated with significant GFP induction as compared to the DMSO control (Fig. 8B). The finding that compounds desilencing the automiG construct in Drosophila cells also desilence the CMV-automiG variant in HeLa cells further validates automiG as a versatile biosensor for miRNA biogenesis and activity.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

Show MeSH
Related in: MedlinePlus