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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-191 duplex and its derivatives. Control siGY441 (A)–(E), wild-type A-type (F)–(J), I-type (K)–(O), or G-type (P)–(T) miR-191 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.
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Figure 5: The results of reporter assays using miR-191 duplex and its derivatives. Control siGY441 (A)–(E), wild-type A-type (F)–(J), I-type (K)–(O), or G-type (P)–(T) miR-191 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.

Mentions: Microarray experiments revealed that the expression of C-target transcripts were downregulated by G-type miRNAs. Moreover, I-type miRNAs could reduce the expression levels of C-target mRNAs, but the reduction levels were not necessarily equivalent to those of G-type miRNAs and the effect of inosine seemed to vary between the miRNA species tested. However, a well-known caveat of microarray experiments is that gene expression may be affected by secondary effects and/or miRNA off-target effects. Therefore, we performed an in vivo luciferase assay in order to confirm the direct effect of each miRNA on their target mRNAs. Two types of reporter plasmids were constructed (Figures 3–5): (i) psiCHECK–SM-target, which contains three tandem repeats of seed-matched (SM) sequences with complementarity to the seed region only (Figures 3–5, upper left panels). (ii) psiCHECK–CM-target with a single complete-matched (CM) sequence corresponding to the full-length mature miRNA, as a positive control (Figures 3–5, upper right panels). The SM or CM target sequences were inserted into the 3′-UTR of the Renilla luciferase gene in a psiCHECK-1 vector (see Materials and Methods section, upper panels of Figures 3–5). In both constructs, uridine or cytidine was introduced into the target sequence at the opposite position (position X in the target sequences of the upper panels in Figures 3–5) of the possible editing site of miRNA, and shown as SM-U-target or SM-C-target, and CM-U-target or CM-C-target, respectively (Figures 3–5). Each of these reporter constructs was co-transfected into human HeLa cells with pGL3-Control encoding firefly luciferase gene and each miRNA, and relative luciferase activity was measured one day after transfection. The results of luciferase reporter assays for all combinations of targets and miRNAs were shown in Figures 3–5, and those of the specific combinations (SM-U-targets and A-type miRNA, SM-C-targets and I-type miRNAs and SM-C-targets and G-type miRNAs) were picked up and compared in Figure 6. All three A-type wild-type miRNA duplexes (miR-376a-2, miR-22 and miR-191 duplexes) reduced the luciferase activity of their SM-U-targets in a dose-dependent manner, but not that of SM-C-targets (Figures 3–6). Consistently, all of the G-type miRNAs reduced the luciferase activity of their SM-C-targets, but not that of SM-U-targets (Figures 3–6). However, the effects of I-type miRNAs were different between miRNA species: the I-type miR-376a-2 duplex did not significantly repress its SM-C-target (Figures 3 and 6A), whereas the I-type miR-22 (Figures 4 and 6B) and miR-191 duplexes (Figures 5 and 6C) significantly reduced the luciferase activity of SM-C-target reporters, albeit at different levels. These results were well correlated with those of the microarray experiment (Figure 2), with the C-target silencing activity of I-type miRNAs in order of miR-191 > miR-22 > miR-376a-2. Finally, we could verify the effectiveness on CM-targets of all A-type, I-type and G-type miRNA duplexes used in this study, indicating that these miRNAs are functional and not defective (Figures 3–5, CM-target).


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-191 duplex and its derivatives. Control siGY441 (A)–(E), wild-type A-type (F)–(J), I-type (K)–(O), or G-type (P)–(T) miR-191 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.
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Related In: Results  -  Collection

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Figure 5: The results of reporter assays using miR-191 duplex and its derivatives. Control siGY441 (A)–(E), wild-type A-type (F)–(J), I-type (K)–(O), or G-type (P)–(T) miR-191 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.
Mentions: Microarray experiments revealed that the expression of C-target transcripts were downregulated by G-type miRNAs. Moreover, I-type miRNAs could reduce the expression levels of C-target mRNAs, but the reduction levels were not necessarily equivalent to those of G-type miRNAs and the effect of inosine seemed to vary between the miRNA species tested. However, a well-known caveat of microarray experiments is that gene expression may be affected by secondary effects and/or miRNA off-target effects. Therefore, we performed an in vivo luciferase assay in order to confirm the direct effect of each miRNA on their target mRNAs. Two types of reporter plasmids were constructed (Figures 3–5): (i) psiCHECK–SM-target, which contains three tandem repeats of seed-matched (SM) sequences with complementarity to the seed region only (Figures 3–5, upper left panels). (ii) psiCHECK–CM-target with a single complete-matched (CM) sequence corresponding to the full-length mature miRNA, as a positive control (Figures 3–5, upper right panels). The SM or CM target sequences were inserted into the 3′-UTR of the Renilla luciferase gene in a psiCHECK-1 vector (see Materials and Methods section, upper panels of Figures 3–5). In both constructs, uridine or cytidine was introduced into the target sequence at the opposite position (position X in the target sequences of the upper panels in Figures 3–5) of the possible editing site of miRNA, and shown as SM-U-target or SM-C-target, and CM-U-target or CM-C-target, respectively (Figures 3–5). Each of these reporter constructs was co-transfected into human HeLa cells with pGL3-Control encoding firefly luciferase gene and each miRNA, and relative luciferase activity was measured one day after transfection. The results of luciferase reporter assays for all combinations of targets and miRNAs were shown in Figures 3–5, and those of the specific combinations (SM-U-targets and A-type miRNA, SM-C-targets and I-type miRNAs and SM-C-targets and G-type miRNAs) were picked up and compared in Figure 6. All three A-type wild-type miRNA duplexes (miR-376a-2, miR-22 and miR-191 duplexes) reduced the luciferase activity of their SM-U-targets in a dose-dependent manner, but not that of SM-C-targets (Figures 3–6). Consistently, all of the G-type miRNAs reduced the luciferase activity of their SM-C-targets, but not that of SM-U-targets (Figures 3–6). However, the effects of I-type miRNAs were different between miRNA species: the I-type miR-376a-2 duplex did not significantly repress its SM-C-target (Figures 3 and 6A), whereas the I-type miR-22 (Figures 4 and 6B) and miR-191 duplexes (Figures 5 and 6C) significantly reduced the luciferase activity of SM-C-target reporters, albeit at different levels. These results were well correlated with those of the microarray experiment (Figure 2), with the C-target silencing activity of I-type miRNAs in order of miR-191 > miR-22 > miR-376a-2. Finally, we could verify the effectiveness on CM-targets of all A-type, I-type and G-type miRNA duplexes used in this study, indicating that these miRNAs are functional and not defective (Figures 3–5, CM-target).

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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