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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.

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The automiG construct is a sensor of Ago2-mediated miRNA silencing.(A) S2R+ cell lines stably transfected with automiG, automiG-Δ1–2, automiG-1–Δ2 and automiG-Δ1–Δ2 constructs were grown for 48 h in the presence or absence of CuSO4, as indicated and GFP expression was analyzed by western blotting. (B) GFP mRNAs are degraded in automiG expressing cells. S2 cells transformed with automiG or automiG-Δ1–Δ2 constructs were grown for 24 h in the presence of CuSO4. Total RNA was extracted and analyzed by northern blot using GFP and RpL32 radiolabeled probes as indicated. (C) Control of dsRNA efficiency. The automiG S2R+ stable cell line was soaked for 48 h with the indicated dsRNA, induced by CuSO4 for 6 additional hours and expression of Dcr-1, Dcr-2, Ago1 and Ago2 was analyzed by western blot. The γ-Tubulin protein was used as a loading control. (D) automiG S2R+ and automiG-Δ1–Δ2 stable cell lines were soaked for 48 h with the indicated dsRNA, induced by CuSO4 for 6 additional hours and GFP expression was analyzed by western blot. The γ-Tubulin protein was used as a loading control. (E) GFP expression from the ubi-automiG transgene in wild type, heterozygous or homozygous ago2414 or dcr2R416X adult flies was analyzed by western blotting. Ponceau staining was used to control equal loading of the lanes. (F) Ago1 RNAi in the patch territory suppressed the Ago1-dependent miR-7 effect in the wings. Wings from ptc-GAL4>UAS-mir-7 adult flies in the presence of the UAS-GFP transgene as a control or of the hairpin transgenes UAS-IR[Ago1], UAS-IR[Ago2] or UAS-IR[pasha] are shown. Overexpression of mir-7 in the patch territory induces a distal wing notch in control flies, that is suppressed by Ago1 and pasha RNAi knockdowns but not by Ago2 RNAi knockdown. (G) GFP (green) expression in wing discs from ptc-GAL4, UASp-automiG/UAS-IR[Ago1] or ptc-GAL4, UASp-automiG/UAS-IR[Ago2] third-instar larvae is shown. Control discs ptc-GAL4, UASp-automiG/+ did not induce a significant GFP signal in control experiments. DAPI staining is shown in red.
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pone-0074296-g002: The automiG construct is a sensor of Ago2-mediated miRNA silencing.(A) S2R+ cell lines stably transfected with automiG, automiG-Δ1–2, automiG-1–Δ2 and automiG-Δ1–Δ2 constructs were grown for 48 h in the presence or absence of CuSO4, as indicated and GFP expression was analyzed by western blotting. (B) GFP mRNAs are degraded in automiG expressing cells. S2 cells transformed with automiG or automiG-Δ1–Δ2 constructs were grown for 24 h in the presence of CuSO4. Total RNA was extracted and analyzed by northern blot using GFP and RpL32 radiolabeled probes as indicated. (C) Control of dsRNA efficiency. The automiG S2R+ stable cell line was soaked for 48 h with the indicated dsRNA, induced by CuSO4 for 6 additional hours and expression of Dcr-1, Dcr-2, Ago1 and Ago2 was analyzed by western blot. The γ-Tubulin protein was used as a loading control. (D) automiG S2R+ and automiG-Δ1–Δ2 stable cell lines were soaked for 48 h with the indicated dsRNA, induced by CuSO4 for 6 additional hours and GFP expression was analyzed by western blot. The γ-Tubulin protein was used as a loading control. (E) GFP expression from the ubi-automiG transgene in wild type, heterozygous or homozygous ago2414 or dcr2R416X adult flies was analyzed by western blotting. Ponceau staining was used to control equal loading of the lanes. (F) Ago1 RNAi in the patch territory suppressed the Ago1-dependent miR-7 effect in the wings. Wings from ptc-GAL4>UAS-mir-7 adult flies in the presence of the UAS-GFP transgene as a control or of the hairpin transgenes UAS-IR[Ago1], UAS-IR[Ago2] or UAS-IR[pasha] are shown. Overexpression of mir-7 in the patch territory induces a distal wing notch in control flies, that is suppressed by Ago1 and pasha RNAi knockdowns but not by Ago2 RNAi knockdown. (G) GFP (green) expression in wing discs from ptc-GAL4, UASp-automiG/UAS-IR[Ago1] or ptc-GAL4, UASp-automiG/UAS-IR[Ago2] third-instar larvae is shown. Control discs ptc-GAL4, UASp-automiG/+ did not induce a significant GFP signal in control experiments. DAPI staining is shown in red.

Mentions: The GFP protein (Fig. 2A) as well as the GFP mRNA (Fig. 2B) were dramatically down-regulated in copper-induced automiG cells as compared to cells transfected with an automiG-Δ1–Δ2 control construct in which both pre-miG-1 and pre-miG-2 sequences are deleted, demonstrating a strong self-silencing of the automiG construct by the reprogrammed miG-1 and miG-2 miRNAs. GFP expression in a cell line transfected with the automiG-1–Δ2 construct in which only the pre-miG2 sequence is deleted was very modest, whereas it was intermediate in a cell line transfected with an automiG-Δ1–2 cell line in which only the pre-miG-1 sequence is deleted (Fig. 2A). Copy numbers of stably transfected constructs may vary between cell lines. Hence, it is possible that GFP expression levels do not accurately reflect the relative strengths of self-silencing of the automiG variants. Nevertheless, the data suggest that miG-1 predominantly associated to Ago2 contributes more to the net silencing of the automiG construct than miG-2 predominantly associated to Ago1. This conclusion is fully supported by the previous finding that the efficiency of the high turn-over Ago2 enzyme exceeds the one of Ago1 by one order of magnitude for silencing targets with perfect base complementarity [21].


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 automiG construct is a sensor of Ago2-mediated miRNA silencing.(A) S2R+ cell lines stably transfected with automiG, automiG-Δ1–2, automiG-1–Δ2 and automiG-Δ1–Δ2 constructs were grown for 48 h in the presence or absence of CuSO4, as indicated and GFP expression was analyzed by western blotting. (B) GFP mRNAs are degraded in automiG expressing cells. S2 cells transformed with automiG or automiG-Δ1–Δ2 constructs were grown for 24 h in the presence of CuSO4. Total RNA was extracted and analyzed by northern blot using GFP and RpL32 radiolabeled probes as indicated. (C) Control of dsRNA efficiency. The automiG S2R+ stable cell line was soaked for 48 h with the indicated dsRNA, induced by CuSO4 for 6 additional hours and expression of Dcr-1, Dcr-2, Ago1 and Ago2 was analyzed by western blot. The γ-Tubulin protein was used as a loading control. (D) automiG S2R+ and automiG-Δ1–Δ2 stable cell lines were soaked for 48 h with the indicated dsRNA, induced by CuSO4 for 6 additional hours and GFP expression was analyzed by western blot. The γ-Tubulin protein was used as a loading control. (E) GFP expression from the ubi-automiG transgene in wild type, heterozygous or homozygous ago2414 or dcr2R416X adult flies was analyzed by western blotting. Ponceau staining was used to control equal loading of the lanes. (F) Ago1 RNAi in the patch territory suppressed the Ago1-dependent miR-7 effect in the wings. Wings from ptc-GAL4>UAS-mir-7 adult flies in the presence of the UAS-GFP transgene as a control or of the hairpin transgenes UAS-IR[Ago1], UAS-IR[Ago2] or UAS-IR[pasha] are shown. Overexpression of mir-7 in the patch territory induces a distal wing notch in control flies, that is suppressed by Ago1 and pasha RNAi knockdowns but not by Ago2 RNAi knockdown. (G) GFP (green) expression in wing discs from ptc-GAL4, UASp-automiG/UAS-IR[Ago1] or ptc-GAL4, UASp-automiG/UAS-IR[Ago2] third-instar larvae is shown. Control discs ptc-GAL4, UASp-automiG/+ did not induce a significant GFP signal in control experiments. DAPI staining is shown in red.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760873&req=5

pone-0074296-g002: The automiG construct is a sensor of Ago2-mediated miRNA silencing.(A) S2R+ cell lines stably transfected with automiG, automiG-Δ1–2, automiG-1–Δ2 and automiG-Δ1–Δ2 constructs were grown for 48 h in the presence or absence of CuSO4, as indicated and GFP expression was analyzed by western blotting. (B) GFP mRNAs are degraded in automiG expressing cells. S2 cells transformed with automiG or automiG-Δ1–Δ2 constructs were grown for 24 h in the presence of CuSO4. Total RNA was extracted and analyzed by northern blot using GFP and RpL32 radiolabeled probes as indicated. (C) Control of dsRNA efficiency. The automiG S2R+ stable cell line was soaked for 48 h with the indicated dsRNA, induced by CuSO4 for 6 additional hours and expression of Dcr-1, Dcr-2, Ago1 and Ago2 was analyzed by western blot. The γ-Tubulin protein was used as a loading control. (D) automiG S2R+ and automiG-Δ1–Δ2 stable cell lines were soaked for 48 h with the indicated dsRNA, induced by CuSO4 for 6 additional hours and GFP expression was analyzed by western blot. The γ-Tubulin protein was used as a loading control. (E) GFP expression from the ubi-automiG transgene in wild type, heterozygous or homozygous ago2414 or dcr2R416X adult flies was analyzed by western blotting. Ponceau staining was used to control equal loading of the lanes. (F) Ago1 RNAi in the patch territory suppressed the Ago1-dependent miR-7 effect in the wings. Wings from ptc-GAL4>UAS-mir-7 adult flies in the presence of the UAS-GFP transgene as a control or of the hairpin transgenes UAS-IR[Ago1], UAS-IR[Ago2] or UAS-IR[pasha] are shown. Overexpression of mir-7 in the patch territory induces a distal wing notch in control flies, that is suppressed by Ago1 and pasha RNAi knockdowns but not by Ago2 RNAi knockdown. (G) GFP (green) expression in wing discs from ptc-GAL4, UASp-automiG/UAS-IR[Ago1] or ptc-GAL4, UASp-automiG/UAS-IR[Ago2] third-instar larvae is shown. Control discs ptc-GAL4, UASp-automiG/+ did not induce a significant GFP signal in control experiments. DAPI staining is shown in red.
Mentions: The GFP protein (Fig. 2A) as well as the GFP mRNA (Fig. 2B) were dramatically down-regulated in copper-induced automiG cells as compared to cells transfected with an automiG-Δ1–Δ2 control construct in which both pre-miG-1 and pre-miG-2 sequences are deleted, demonstrating a strong self-silencing of the automiG construct by the reprogrammed miG-1 and miG-2 miRNAs. GFP expression in a cell line transfected with the automiG-1–Δ2 construct in which only the pre-miG2 sequence is deleted was very modest, whereas it was intermediate in a cell line transfected with an automiG-Δ1–2 cell line in which only the pre-miG-1 sequence is deleted (Fig. 2A). Copy numbers of stably transfected constructs may vary between cell lines. Hence, it is possible that GFP expression levels do not accurately reflect the relative strengths of self-silencing of the automiG variants. Nevertheless, the data suggest that miG-1 predominantly associated to Ago2 contributes more to the net silencing of the automiG construct than miG-2 predominantly associated to Ago1. This conclusion is fully supported by the previous finding that the efficiency of the high turn-over Ago2 enzyme exceeds the one of Ago1 by one order of magnitude for silencing targets with perfect base complementarity [21].

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