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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 expresses miRNAs targeted to GFP sequences.(A) Schemes of the miR5-6.1-GFP and automiG constructs. (B) Folded sequences of miR-5, miR-6.1, miG-1 and miG-2 precursors in miR5-6.1-GFP or automiG constructs. Positions of mature miR-5 and miR-6.1 sequences are indicated with blue bars. Positions of mature miG-1 and miG-2 sequences are indicated with green bars. Positions of miG-1* and miG-2* sequences are indicated with grey bars. (C) S2R+ cell lines stably transfected with miR5-6.1-GFP or automiG were grown for 48 h in the presence or absence of CuSO4. Total RNA from these cells was analyzed in northern blot using the indicated radiolabeled probes. U6 RNA was used as a loading control. (D) Solexa sequencing of small RNA libraries prepared from S2R+ cells or transgenic flies stably transfected with the ubi-automiG construct. The oxidized library was generated from small RNA treated by NaIO4 before adapter ligation. For each library, the total number of reads of Drosophila miRNAs (all) is indicated, as well as the number of reads of bantam, miG-1, miG-2 and of the corresponding miR* species. Frequencies are expressed relatively to the total number of miRNA reads in the libraries. (E) S2 cells stably transfected with automiG and a construct expressing a tagged Ago2-HA protein were grown for 48 h in the presence of CuSO4. A total extract from these cells was split and immunoprecipitated using anti-Ago1 (Ago1-IP) or anti-HA (Ago-2-IP) antibodies. Protein content of input (I), supernatant (S) and bound fractions (B) was analyzed by western blot using the indicated antibodies. RNA was extracted from I, S and B fractions and analyzed by northern blot using miG-1, miG-2, miG-1*, miG-2* and bantam radiolabeled probes.
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pone-0074296-g001: The automiG construct expresses miRNAs targeted to GFP sequences.(A) Schemes of the miR5-6.1-GFP and automiG constructs. (B) Folded sequences of miR-5, miR-6.1, miG-1 and miG-2 precursors in miR5-6.1-GFP or automiG constructs. Positions of mature miR-5 and miR-6.1 sequences are indicated with blue bars. Positions of mature miG-1 and miG-2 sequences are indicated with green bars. Positions of miG-1* and miG-2* sequences are indicated with grey bars. (C) S2R+ cell lines stably transfected with miR5-6.1-GFP or automiG were grown for 48 h in the presence or absence of CuSO4. Total RNA from these cells was analyzed in northern blot using the indicated radiolabeled probes. U6 RNA was used as a loading control. (D) Solexa sequencing of small RNA libraries prepared from S2R+ cells or transgenic flies stably transfected with the ubi-automiG construct. The oxidized library was generated from small RNA treated by NaIO4 before adapter ligation. For each library, the total number of reads of Drosophila miRNAs (all) is indicated, as well as the number of reads of bantam, miG-1, miG-2 and of the corresponding miR* species. Frequencies are expressed relatively to the total number of miRNA reads in the libraries. (E) S2 cells stably transfected with automiG and a construct expressing a tagged Ago2-HA protein were grown for 48 h in the presence of CuSO4. A total extract from these cells was split and immunoprecipitated using anti-Ago1 (Ago1-IP) or anti-HA (Ago-2-IP) antibodies. Protein content of input (I), supernatant (S) and bound fractions (B) was analyzed by western blot using the indicated antibodies. RNA was extracted from I, S and B fractions and analyzed by northern blot using miG-1, miG-2, miG-1*, miG-2* and bantam radiolabeled probes.

Mentions: A Gateway pENTR-3C vector (Invitrogen) was engineered to give rise to pENTR-3C_miR5-miR6. This construct includes the exon2-intron2-exon3 region of the RpL17 gene fused to the GFP coding sequences. We replaced a 262 bp region from the RpL17 intron by a 262 bp genomic region containing mir-5 and mi-6-1 in which EcoRI, SphI, HindIII and ClaI sites were introduced to facilitate subsequent mir substitution. (plasmid map available upon request). A pENTR-3C_miG1_miG2 vector was then produced by replacing the EcoRI-mir-5-SphI and HindIII-mir-6-1-ClaI fragments in pENTR-3C_miR5-miR6 by EcoRI-miG1-SphI and HindIII-miG2-ClaI sequences, as depicted in Fig. 1A. Derivative constructs pENTR-3C_Δ1-miG2, pENTR-3C_Δ1–Δ2 and pENTR-3C_miG1-Δ2 were generated by restriction-mediated deletion of miG1, miG2 or both miG1 and miG2 segments. Finally, appropriate pENTR derivative vectors were recombined with the Gateway pDEST-48 destination vector for metallothionein promoter driven expression in Drosophila cells (Invitrogen) to give rise to the miR5-6.1-GFP, automiG, automiG-Δ1–2, automiG-1–Δ2 and automiG-Δ1–Δ2 constructs. The ubiquitin-automiG construct variant used for Drosophila transgenesis was generated by recombining the pENTR-3C_miG1–miG2 vector with the destination vector pUWG (a kind gift from Clara Moch and Jean-René Huynh) described at the Drosophila Genome Ressource Center (https://dgrc.cgb.indiana.edu/). pCI-neo mammalian expression vector (Promega®) was used as backbone vector to generate a Gateway compatible destination vector using the Gateway® Vector Conversion System. pCI-neo destination vector (a gift from Yves Jacob, Pasteur Institute) was then recombined with the previously described pENTR-3C_miG1_miG2 to give rise to the CMV-automiG vector driven automiG expression in mammalian cells. Sequences of all constructs were verified and are available upon request.


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 expresses miRNAs targeted to GFP sequences.(A) Schemes of the miR5-6.1-GFP and automiG constructs. (B) Folded sequences of miR-5, miR-6.1, miG-1 and miG-2 precursors in miR5-6.1-GFP or automiG constructs. Positions of mature miR-5 and miR-6.1 sequences are indicated with blue bars. Positions of mature miG-1 and miG-2 sequences are indicated with green bars. Positions of miG-1* and miG-2* sequences are indicated with grey bars. (C) S2R+ cell lines stably transfected with miR5-6.1-GFP or automiG were grown for 48 h in the presence or absence of CuSO4. Total RNA from these cells was analyzed in northern blot using the indicated radiolabeled probes. U6 RNA was used as a loading control. (D) Solexa sequencing of small RNA libraries prepared from S2R+ cells or transgenic flies stably transfected with the ubi-automiG construct. The oxidized library was generated from small RNA treated by NaIO4 before adapter ligation. For each library, the total number of reads of Drosophila miRNAs (all) is indicated, as well as the number of reads of bantam, miG-1, miG-2 and of the corresponding miR* species. Frequencies are expressed relatively to the total number of miRNA reads in the libraries. (E) S2 cells stably transfected with automiG and a construct expressing a tagged Ago2-HA protein were grown for 48 h in the presence of CuSO4. A total extract from these cells was split and immunoprecipitated using anti-Ago1 (Ago1-IP) or anti-HA (Ago-2-IP) antibodies. Protein content of input (I), supernatant (S) and bound fractions (B) was analyzed by western blot using the indicated antibodies. RNA was extracted from I, S and B fractions and analyzed by northern blot using miG-1, miG-2, miG-1*, miG-2* and bantam radiolabeled probes.
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getmorefigures.php?uid=PMC3760873&req=5

pone-0074296-g001: The automiG construct expresses miRNAs targeted to GFP sequences.(A) Schemes of the miR5-6.1-GFP and automiG constructs. (B) Folded sequences of miR-5, miR-6.1, miG-1 and miG-2 precursors in miR5-6.1-GFP or automiG constructs. Positions of mature miR-5 and miR-6.1 sequences are indicated with blue bars. Positions of mature miG-1 and miG-2 sequences are indicated with green bars. Positions of miG-1* and miG-2* sequences are indicated with grey bars. (C) S2R+ cell lines stably transfected with miR5-6.1-GFP or automiG were grown for 48 h in the presence or absence of CuSO4. Total RNA from these cells was analyzed in northern blot using the indicated radiolabeled probes. U6 RNA was used as a loading control. (D) Solexa sequencing of small RNA libraries prepared from S2R+ cells or transgenic flies stably transfected with the ubi-automiG construct. The oxidized library was generated from small RNA treated by NaIO4 before adapter ligation. For each library, the total number of reads of Drosophila miRNAs (all) is indicated, as well as the number of reads of bantam, miG-1, miG-2 and of the corresponding miR* species. Frequencies are expressed relatively to the total number of miRNA reads in the libraries. (E) S2 cells stably transfected with automiG and a construct expressing a tagged Ago2-HA protein were grown for 48 h in the presence of CuSO4. A total extract from these cells was split and immunoprecipitated using anti-Ago1 (Ago1-IP) or anti-HA (Ago-2-IP) antibodies. Protein content of input (I), supernatant (S) and bound fractions (B) was analyzed by western blot using the indicated antibodies. RNA was extracted from I, S and B fractions and analyzed by northern blot using miG-1, miG-2, miG-1*, miG-2* and bantam radiolabeled probes.
Mentions: A Gateway pENTR-3C vector (Invitrogen) was engineered to give rise to pENTR-3C_miR5-miR6. This construct includes the exon2-intron2-exon3 region of the RpL17 gene fused to the GFP coding sequences. We replaced a 262 bp region from the RpL17 intron by a 262 bp genomic region containing mir-5 and mi-6-1 in which EcoRI, SphI, HindIII and ClaI sites were introduced to facilitate subsequent mir substitution. (plasmid map available upon request). A pENTR-3C_miG1_miG2 vector was then produced by replacing the EcoRI-mir-5-SphI and HindIII-mir-6-1-ClaI fragments in pENTR-3C_miR5-miR6 by EcoRI-miG1-SphI and HindIII-miG2-ClaI sequences, as depicted in Fig. 1A. Derivative constructs pENTR-3C_Δ1-miG2, pENTR-3C_Δ1–Δ2 and pENTR-3C_miG1-Δ2 were generated by restriction-mediated deletion of miG1, miG2 or both miG1 and miG2 segments. Finally, appropriate pENTR derivative vectors were recombined with the Gateway pDEST-48 destination vector for metallothionein promoter driven expression in Drosophila cells (Invitrogen) to give rise to the miR5-6.1-GFP, automiG, automiG-Δ1–2, automiG-1–Δ2 and automiG-Δ1–Δ2 constructs. The ubiquitin-automiG construct variant used for Drosophila transgenesis was generated by recombining the pENTR-3C_miG1–miG2 vector with the destination vector pUWG (a kind gift from Clara Moch and Jean-René Huynh) described at the Drosophila Genome Ressource Center (https://dgrc.cgb.indiana.edu/). pCI-neo mammalian expression vector (Promega®) was used as backbone vector to generate a Gateway compatible destination vector using the Gateway® Vector Conversion System. pCI-neo destination vector (a gift from Yves Jacob, Pasteur Institute) was then recombined with the previously described pENTR-3C_miG1_miG2 to give rise to the CMV-automiG vector driven automiG expression in mammalian cells. Sequences of all constructs were verified and are available upon request.

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