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A-to-I editing in the miRNA seed region regulates target mRNA selection and silencing efficiency.

Kume H, Hino K, Galipon J, Ui-Tei K - Nucleic Acids Res. (2014)

Bottom Line: Hydrolytic deamination of adenosine to inosine (A-to-I) by adenosine deaminases acting on RNA (ADARs) is a post-transcriptional modification which results in a discrepancy between genomic DNA and the transcribed RNA sequence, thus contributing to the diversity of the transcriptome.The difference in base-pairing stability, deduced by melting temperature measurements, between seed-target duplexes containing either C:G or I:C pairs may account for the observed silencing efficiency.These findings unequivocally show that C:G and I:C pairs are biologically different in terms of gene expression regulation by miRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

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The results of reporter assays using miR-376a-2 duplex and its derivatives. The upper panels indicate the schematic structures of psiCHECK-SM-target and psiCHECK-CM-target constructs containing three tandem repeats of SM-target sequences and a CM-target sequence, respectively, in the 3′ UTR of Renilla luciferase gene of psiCHECK vector. Position Y indicates the possible editing site in the mRNA, and position X in the SM- or CM-target sequence indicates the position opposite to the the possible editing site of miRNA. Control siGY441 (A)–(E), wild-type A-type (F)–(J), I-type (K)–(O), or G-type (P)–(T) miR-376a-2 duplex was transfected with control psiCHECK-1 (A), (F), (K), (P), psiCHECK-SM-U-target (B), (G), (L), (Q), psiCHECK-SM-C-target (C), (H), (M), (R), psiCHECK-CM-U-target (D), (I), (N), (S), or psiCHECK-CM-C-target (E), (J), (O), (T), and pGL3-Control firefly luciferase expression construct were simultaneously transfected into HeLa cells. Relative luciferase activity was measured one day after transfection. Each miRNA was transfected at 0.05, 0.5, 5 and 50 nM, respectively. Complementarity between transfected miRNA and target construct is shown at top of each figure, and the possible editing sites were highlighted. Bar indicates the standard deviation of triplicated samples.
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Figure 3: The results of reporter assays using miR-376a-2 duplex and its derivatives. The upper panels indicate the schematic structures of psiCHECK-SM-target and psiCHECK-CM-target constructs containing three tandem repeats of SM-target sequences and a CM-target sequence, respectively, in the 3′ UTR of Renilla luciferase gene of psiCHECK vector. Position Y indicates the possible editing site in the mRNA, and position X in the SM- or CM-target sequence indicates the position opposite to the the possible editing site of miRNA. Control siGY441 (A)–(E), wild-type A-type (F)–(J), I-type (K)–(O), or G-type (P)–(T) miR-376a-2 duplex was transfected with control psiCHECK-1 (A), (F), (K), (P), psiCHECK-SM-U-target (B), (G), (L), (Q), psiCHECK-SM-C-target (C), (H), (M), (R), psiCHECK-CM-U-target (D), (I), (N), (S), or psiCHECK-CM-C-target (E), (J), (O), (T), and pGL3-Control firefly luciferase expression construct were simultaneously transfected into HeLa cells. Relative luciferase activity was measured one day after transfection. Each miRNA was transfected at 0.05, 0.5, 5 and 50 nM, respectively. Complementarity between transfected miRNA and target construct is shown at top of each figure, and the possible editing sites were highlighted. Bar indicates the standard deviation of triplicated samples.

Mentions: The results of reporter assays using miR-22 duplex and its derivatives. Control siGY441 (A)–(E), wild-type A-type (F)–(J), I-type (K)–(O), or G-type (P)–(T) miR-22 duplex was transfected with control psiCHECK-1 (A), (F), (K), (P), psiCHECK-SM-U-target (B), (G), (L), (Q), psiCHECK-SM-C-target (C), (H), (M), (R), psiCHECK-CM-U-target (D), (I), (N), (S), or psiCHECK-CM-C-target (E), (J), (O), (T), and pGL3-Control firefly luciferase expression construct were simultaneously transfected into HeLa cells. Other detailed descriptions about this figure are same as those of Figure 3.


A-to-I editing in the miRNA seed region regulates target mRNA selection and silencing efficiency.

Kume H, Hino K, Galipon J, Ui-Tei K - Nucleic Acids Res. (2014)

The results of reporter assays using miR-376a-2 duplex and its derivatives. The upper panels indicate the schematic structures of psiCHECK-SM-target and psiCHECK-CM-target constructs containing three tandem repeats of SM-target sequences and a CM-target sequence, respectively, in the 3′ UTR of Renilla luciferase gene of psiCHECK vector. Position Y indicates the possible editing site in the mRNA, and position X in the SM- or CM-target sequence indicates the position opposite to the the possible editing site of miRNA. Control siGY441 (A)–(E), wild-type A-type (F)–(J), I-type (K)–(O), or G-type (P)–(T) miR-376a-2 duplex was transfected with control psiCHECK-1 (A), (F), (K), (P), psiCHECK-SM-U-target (B), (G), (L), (Q), psiCHECK-SM-C-target (C), (H), (M), (R), psiCHECK-CM-U-target (D), (I), (N), (S), or psiCHECK-CM-C-target (E), (J), (O), (T), and pGL3-Control firefly luciferase expression construct were simultaneously transfected into HeLa cells. Relative luciferase activity was measured one day after transfection. Each miRNA was transfected at 0.05, 0.5, 5 and 50 nM, respectively. Complementarity between transfected miRNA and target construct is shown at top of each figure, and the possible editing sites were highlighted. Bar indicates the standard deviation of triplicated samples.
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Related In: Results  -  Collection

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Show All Figures
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Figure 3: The results of reporter assays using miR-376a-2 duplex and its derivatives. The upper panels indicate the schematic structures of psiCHECK-SM-target and psiCHECK-CM-target constructs containing three tandem repeats of SM-target sequences and a CM-target sequence, respectively, in the 3′ UTR of Renilla luciferase gene of psiCHECK vector. Position Y indicates the possible editing site in the mRNA, and position X in the SM- or CM-target sequence indicates the position opposite to the the possible editing site of miRNA. Control siGY441 (A)–(E), wild-type A-type (F)–(J), I-type (K)–(O), or G-type (P)–(T) miR-376a-2 duplex was transfected with control psiCHECK-1 (A), (F), (K), (P), psiCHECK-SM-U-target (B), (G), (L), (Q), psiCHECK-SM-C-target (C), (H), (M), (R), psiCHECK-CM-U-target (D), (I), (N), (S), or psiCHECK-CM-C-target (E), (J), (O), (T), and pGL3-Control firefly luciferase expression construct were simultaneously transfected into HeLa cells. Relative luciferase activity was measured one day after transfection. Each miRNA was transfected at 0.05, 0.5, 5 and 50 nM, respectively. Complementarity between transfected miRNA and target construct is shown at top of each figure, and the possible editing sites were highlighted. Bar indicates the standard deviation of triplicated samples.
Mentions: The results of reporter assays using miR-22 duplex and its derivatives. Control siGY441 (A)–(E), wild-type A-type (F)–(J), I-type (K)–(O), or G-type (P)–(T) miR-22 duplex was transfected with control psiCHECK-1 (A), (F), (K), (P), psiCHECK-SM-U-target (B), (G), (L), (Q), psiCHECK-SM-C-target (C), (H), (M), (R), psiCHECK-CM-U-target (D), (I), (N), (S), or psiCHECK-CM-C-target (E), (J), (O), (T), and pGL3-Control firefly luciferase expression construct were simultaneously transfected into HeLa cells. Other detailed descriptions about this figure are same as those of Figure 3.

Bottom Line: Hydrolytic deamination of adenosine to inosine (A-to-I) by adenosine deaminases acting on RNA (ADARs) is a post-transcriptional modification which results in a discrepancy between genomic DNA and the transcribed RNA sequence, thus contributing to the diversity of the transcriptome.The difference in base-pairing stability, deduced by melting temperature measurements, between seed-target duplexes containing either C:G or I:C pairs may account for the observed silencing efficiency.These findings unequivocally show that C:G and I:C pairs are biologically different in terms of gene expression regulation by miRNAs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan Faculty of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.

Show MeSH