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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-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.
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Figure 4: 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.

Mentions: RNA oligonucleotides corresponding to the 5p and 3p strands of mature miRNA duplexes (21–24-nt in length) and 7-mer oligonucleotides corresponding to the mRNA binding sequence complementary to the active seed (nucleotides 2–8) were chemically synthesized (Sigma or Genepharma) in accordance with the sequences registered in the miRBase and annealed to form either 5p:3p mature miRNA duplexes and seed:mRNA binding site duplexes (9). In addition, the same oligonucleotides containing either inosine (I-type) or guanosine (G-type) instead of adenosine (A-type) at the possible editing sites were also synthesized. The miRNA strand with wild-type adenosine, inosine and guanosine at the editing site are referred to as miRNA-A, -I and -G, respectively. Both strands of miRNAs or seed duplexes were mixed to a 1:1 ratio in a solution of 10 mM NaCl and 20 mM Tris–HCl (pH 7.5), and annealed by incubation at 95ΒΊC for 5 min followed by cooling down to room temperature. The annealed miRNA duplexes containing either adenosine, inosine or guanosine at the editing sites are referred to as A-type, I-type and G-type miRNAs, respectively. The sequence of the synthetic mature miRNAs (miR-376a-2, miR-22, miR-191) and their structures are shown in Figure 1, those of the seed RNA duplexes are shown in Figure 4. siGY441 with a sequence unrelated to the Renilla luciferase gene was used as a negative control. Duplex formation was verified by electrophoresis on a 15% polyacrylamide gel in Tris Borate EDTA (TBE) buffer.


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-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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150774&req=5

Figure 4: 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.
Mentions: RNA oligonucleotides corresponding to the 5p and 3p strands of mature miRNA duplexes (21–24-nt in length) and 7-mer oligonucleotides corresponding to the mRNA binding sequence complementary to the active seed (nucleotides 2–8) were chemically synthesized (Sigma or Genepharma) in accordance with the sequences registered in the miRBase and annealed to form either 5p:3p mature miRNA duplexes and seed:mRNA binding site duplexes (9). In addition, the same oligonucleotides containing either inosine (I-type) or guanosine (G-type) instead of adenosine (A-type) at the possible editing sites were also synthesized. The miRNA strand with wild-type adenosine, inosine and guanosine at the editing site are referred to as miRNA-A, -I and -G, respectively. Both strands of miRNAs or seed duplexes were mixed to a 1:1 ratio in a solution of 10 mM NaCl and 20 mM Tris–HCl (pH 7.5), and annealed by incubation at 95ΒΊC for 5 min followed by cooling down to room temperature. The annealed miRNA duplexes containing either adenosine, inosine or guanosine at the editing sites are referred to as A-type, I-type and G-type miRNAs, respectively. The sequence of the synthetic mature miRNAs (miR-376a-2, miR-22, miR-191) and their structures are shown in Figure 1, those of the seed RNA duplexes are shown in Figure 4. siGY441 with a sequence unrelated to the Renilla luciferase gene was used as a negative control. Duplex formation was verified by electrophoresis on a 15% polyacrylamide gel in Tris Borate EDTA (TBE) buffer.

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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