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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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Microarray and real-time PCR analysis of the expression levels of target transcripts with seed complementary sequences. (A) Schematic model describing the analysis workflow for microarray data shown in Supplementary Figures S1–S3. (1) MA plot: the vertical bar indicates the mean log2 of signal intensities relative to those of mock transfection (M value), and the horizontal bar indicates the averaged log10 signal intensities of mock and miRNA transfections (A value). The red plots show seed-matched transcripts and the black plots show background transcripts. (2) Cumulative distribution: the horizontal axis indicates the ‘M value’, and the vertical axis is the cumulative fraction of mRNAs. The red line indicates the cumulative curve of seed-matched transcripts, and the the black line that of background transcripts. The gray area indicates the fold-change in the expression of seed-matched transcripts compared to those of background transcripts. Each of miR376a-2 (B) and (C), miR-22 (D) and (E) and miR-191 (F) and (G) duplexes were transfected into HeLa cells, and the changes in expression levels of transcripts with U-target (B), (D), (F) or C-target (C), (E), (G) sequences were analyzed by microarray. The complementarity between seed region and target mRNAs were shown in left panel in A–F. In the target sequence, N indicates any given nucleotide. In each experiment, three types of miRNAs (A-type, I-type, G-type) was respectively used. Fold-change indicates the difference of gene expression levels between mean log2 of signal intensities of each miRNA targets and background genes. The expression levels of three target mRNAs of miR-376a-3p (Rad23A, CKAP4 and SEL1L3) (H) or miR-22–3p (EPS15, SLC7A1 and TTYH3) (I) by mock transfection (black bar), I-type miRNA (red bar), or G-type miRNA (yellow bar) were measured by qRT-PCR, and compared with the microarray data (each of black, red, or yellow diagonal bar). Note that the number of seed-complementary target sites ranged from one to four. The seed regions and their complementary site are surrounded by a box, and the possible editing sites were highlighted in orange. In (H) and (I), positions relative to the 5′ end of mRNA were shown. In (H) and (I), a bar indicates the standard deviation calculated from three independent experiments.
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Figure 2: Microarray and real-time PCR analysis of the expression levels of target transcripts with seed complementary sequences. (A) Schematic model describing the analysis workflow for microarray data shown in Supplementary Figures S1–S3. (1) MA plot: the vertical bar indicates the mean log2 of signal intensities relative to those of mock transfection (M value), and the horizontal bar indicates the averaged log10 signal intensities of mock and miRNA transfections (A value). The red plots show seed-matched transcripts and the black plots show background transcripts. (2) Cumulative distribution: the horizontal axis indicates the ‘M value’, and the vertical axis is the cumulative fraction of mRNAs. The red line indicates the cumulative curve of seed-matched transcripts, and the the black line that of background transcripts. The gray area indicates the fold-change in the expression of seed-matched transcripts compared to those of background transcripts. Each of miR376a-2 (B) and (C), miR-22 (D) and (E) and miR-191 (F) and (G) duplexes were transfected into HeLa cells, and the changes in expression levels of transcripts with U-target (B), (D), (F) or C-target (C), (E), (G) sequences were analyzed by microarray. The complementarity between seed region and target mRNAs were shown in left panel in A–F. In the target sequence, N indicates any given nucleotide. In each experiment, three types of miRNAs (A-type, I-type, G-type) was respectively used. Fold-change indicates the difference of gene expression levels between mean log2 of signal intensities of each miRNA targets and background genes. The expression levels of three target mRNAs of miR-376a-3p (Rad23A, CKAP4 and SEL1L3) (H) or miR-22–3p (EPS15, SLC7A1 and TTYH3) (I) by mock transfection (black bar), I-type miRNA (red bar), or G-type miRNA (yellow bar) were measured by qRT-PCR, and compared with the microarray data (each of black, red, or yellow diagonal bar). Note that the number of seed-complementary target sites ranged from one to four. The seed regions and their complementary site are surrounded by a box, and the possible editing sites were highlighted in orange. In (H) and (I), positions relative to the 5′ end of mRNA were shown. In (H) and (I), a bar indicates the standard deviation calculated from three independent experiments.

Mentions: To analyze miRNA-induced global changes in gene expression, mRNAs were divided into four groups based on the presence in their 3′-UTRs of at least one sequence complementary to the seed region containing either A, U, G or C at the possible editing site, respectively. The mRNAs that belonged to more than one of these groups, and those with 3′-UTR complementarity to the dead seed were eliminated to select for targets that may be downregulated by only one type of seed sequence. These separate groups are subsequently referred to as (I) U-target, (II) A-target, (III) C-target, (IV) G-target mRNAs (see Supplementary Figures S1–S3). Consequently, A-type miRNAs are expected to specifically downregulate ‘U-target’ genes, whereas the I-type and G-type miRNAs should downregulate ‘C-target’ genes. The background constitutes of all mRNAs—excluding dead seed targets—that are outside of each individual group. As a result, the background differs slightly between the four groups. The procedure for microarray data analysis is illustrated in Figure 2A. At first, we made an MA plots showing the mean log2 of signal intensities relative to those of mock transfection (M value) and the averaged log10 signal intensities of mock and miRNA transfections (A value), and plotted these values as the vertical and horizontal bars, respectively (Figure 2A(1)). To facilitate understanding, the MA plot was then converted to the cumulative distribution (Figure 2A(2)), in which the horizontal axis indicates the ‘M value’ and the vertical axis is the cumulative fraction of mRNAs. The mean log2 of signal intensities relative to those of mock transfection was calculated for each group of mRNAs and the results are shown as MA plots and cumulative distributions (Supplementary Figures S1–S3). Furthermore, to simplify the results, the ‘area’ between seed-matched mRNAs (red curve in Figure 2A(2)) and background RNAs (black curve in Figure 2A(2)) was calculated and shown as the ‘fold-change’ in Figure 2B–G, with positive values meaning that the target genes are generally more inhibited than background. Furthermore, the Wilcoxon rank-sum test was used to assess whether or not the target genes and background distributions are significantly different (p < 1×10−2).


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)

Microarray and real-time PCR analysis of the expression levels of target transcripts with seed complementary sequences. (A) Schematic model describing the analysis workflow for microarray data shown in Supplementary Figures S1–S3. (1) MA plot: the vertical bar indicates the mean log2 of signal intensities relative to those of mock transfection (M value), and the horizontal bar indicates the averaged log10 signal intensities of mock and miRNA transfections (A value). The red plots show seed-matched transcripts and the black plots show background transcripts. (2) Cumulative distribution: the horizontal axis indicates the ‘M value’, and the vertical axis is the cumulative fraction of mRNAs. The red line indicates the cumulative curve of seed-matched transcripts, and the the black line that of background transcripts. The gray area indicates the fold-change in the expression of seed-matched transcripts compared to those of background transcripts. Each of miR376a-2 (B) and (C), miR-22 (D) and (E) and miR-191 (F) and (G) duplexes were transfected into HeLa cells, and the changes in expression levels of transcripts with U-target (B), (D), (F) or C-target (C), (E), (G) sequences were analyzed by microarray. The complementarity between seed region and target mRNAs were shown in left panel in A–F. In the target sequence, N indicates any given nucleotide. In each experiment, three types of miRNAs (A-type, I-type, G-type) was respectively used. Fold-change indicates the difference of gene expression levels between mean log2 of signal intensities of each miRNA targets and background genes. The expression levels of three target mRNAs of miR-376a-3p (Rad23A, CKAP4 and SEL1L3) (H) or miR-22–3p (EPS15, SLC7A1 and TTYH3) (I) by mock transfection (black bar), I-type miRNA (red bar), or G-type miRNA (yellow bar) were measured by qRT-PCR, and compared with the microarray data (each of black, red, or yellow diagonal bar). Note that the number of seed-complementary target sites ranged from one to four. The seed regions and their complementary site are surrounded by a box, and the possible editing sites were highlighted in orange. In (H) and (I), positions relative to the 5′ end of mRNA were shown. In (H) and (I), a bar indicates the standard deviation calculated from three independent experiments.
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Figure 2: Microarray and real-time PCR analysis of the expression levels of target transcripts with seed complementary sequences. (A) Schematic model describing the analysis workflow for microarray data shown in Supplementary Figures S1–S3. (1) MA plot: the vertical bar indicates the mean log2 of signal intensities relative to those of mock transfection (M value), and the horizontal bar indicates the averaged log10 signal intensities of mock and miRNA transfections (A value). The red plots show seed-matched transcripts and the black plots show background transcripts. (2) Cumulative distribution: the horizontal axis indicates the ‘M value’, and the vertical axis is the cumulative fraction of mRNAs. The red line indicates the cumulative curve of seed-matched transcripts, and the the black line that of background transcripts. The gray area indicates the fold-change in the expression of seed-matched transcripts compared to those of background transcripts. Each of miR376a-2 (B) and (C), miR-22 (D) and (E) and miR-191 (F) and (G) duplexes were transfected into HeLa cells, and the changes in expression levels of transcripts with U-target (B), (D), (F) or C-target (C), (E), (G) sequences were analyzed by microarray. The complementarity between seed region and target mRNAs were shown in left panel in A–F. In the target sequence, N indicates any given nucleotide. In each experiment, three types of miRNAs (A-type, I-type, G-type) was respectively used. Fold-change indicates the difference of gene expression levels between mean log2 of signal intensities of each miRNA targets and background genes. The expression levels of three target mRNAs of miR-376a-3p (Rad23A, CKAP4 and SEL1L3) (H) or miR-22–3p (EPS15, SLC7A1 and TTYH3) (I) by mock transfection (black bar), I-type miRNA (red bar), or G-type miRNA (yellow bar) were measured by qRT-PCR, and compared with the microarray data (each of black, red, or yellow diagonal bar). Note that the number of seed-complementary target sites ranged from one to four. The seed regions and their complementary site are surrounded by a box, and the possible editing sites were highlighted in orange. In (H) and (I), positions relative to the 5′ end of mRNA were shown. In (H) and (I), a bar indicates the standard deviation calculated from three independent experiments.
Mentions: To analyze miRNA-induced global changes in gene expression, mRNAs were divided into four groups based on the presence in their 3′-UTRs of at least one sequence complementary to the seed region containing either A, U, G or C at the possible editing site, respectively. The mRNAs that belonged to more than one of these groups, and those with 3′-UTR complementarity to the dead seed were eliminated to select for targets that may be downregulated by only one type of seed sequence. These separate groups are subsequently referred to as (I) U-target, (II) A-target, (III) C-target, (IV) G-target mRNAs (see Supplementary Figures S1–S3). Consequently, A-type miRNAs are expected to specifically downregulate ‘U-target’ genes, whereas the I-type and G-type miRNAs should downregulate ‘C-target’ genes. The background constitutes of all mRNAs—excluding dead seed targets—that are outside of each individual group. As a result, the background differs slightly between the four groups. The procedure for microarray data analysis is illustrated in Figure 2A. At first, we made an MA plots showing the mean log2 of signal intensities relative to those of mock transfection (M value) and the averaged log10 signal intensities of mock and miRNA transfections (A value), and plotted these values as the vertical and horizontal bars, respectively (Figure 2A(1)). To facilitate understanding, the MA plot was then converted to the cumulative distribution (Figure 2A(2)), in which the horizontal axis indicates the ‘M value’ and the vertical axis is the cumulative fraction of mRNAs. The mean log2 of signal intensities relative to those of mock transfection was calculated for each group of mRNAs and the results are shown as MA plots and cumulative distributions (Supplementary Figures S1–S3). Furthermore, to simplify the results, the ‘area’ between seed-matched mRNAs (red curve in Figure 2A(2)) and background RNAs (black curve in Figure 2A(2)) was calculated and shown as the ‘fold-change’ in Figure 2B–G, with positive values meaning that the target genes are generally more inhibited than background. Furthermore, the Wilcoxon rank-sum test was used to assess whether or not the target genes and background distributions are significantly different (p < 1×10−2).

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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Related in: MedlinePlus