June 25, 2014;
Conserved microRNA editing in mammalian evolution, development and disease.
Mammalian microRNAs (miRNAs) are sometimes subject to adenosine-to-inosine RNA editing, which can lead to dramatic changes in miRNA target specificity or expression levels. However, although a few miRNAs are known to be edited at identical positions in human and mouse, the evolution of miRNA editing has not been investigated in detail. In this study, we identify conserved miRNA editing events in a range of mammalian and non-mammalian species. We demonstrate deep
conservation of several site-specific miRNA editing events, including two that date back to the common ancestor of mammals and bony fishes some 450 million years ago. We also find evidence of a recent expansion of an edited miRNA family in placental mammals and show that editing of these miRNAs is associated with changes in target mRNA expression during primate development and aging. While global patterns of miRNA editing tend to be conserved across species, we observe substantial variation in editing frequencies depending on tissue
, age and disease state: editing is more frequent in neural tissues compared to heart
; in older compared to younger individuals; and in samples from healthy tissues compared to tumors, which together suggests that miRNA editing might be associated with a reduced rate of cell proliferation. Our results show that site-specific miRNA editing is an evolutionarily conserved mechanism, which increases the functional diversity of mammalian miRNA transcriptomes. Furthermore, we find that although miRNA editing is rare compared to editing of long RNAs, miRNAs are greatly overrepresented among conserved editing targets.
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Figure 1. Conservation of miRNA editing in vertebrates. The identifier of the edited miRNA and the position at which editing occurs are indicated on the left. Observed miRNA editing with a frequency of >1% is indicated as blue boxes for the species in the core set (Table 1) and as dark green boxes for the 10 additional species. For completeness, cases where A-to-G mismatches were present at a frequency <1% (‘trace reads’) are shown as light green boxes, although it should be noted that such trace events cannot be readily distinguished from sequencing errors. Gray shading indicates that the presence of miRNA editing could not be assessed due to a lack of sequencing reads for the miRNA in question (which might be explained by the rigorous filtering criteria, lack of expression in the investigated samples or absence of the miRNA in the genome). An asterisk following the species name indicates that no quality scores were available for the species in question. Divergence times were taken from the TimeTree database . Note that the analysis presented here was performed using a stringent detection algorithm to avoid false positives; editing of a given miRNA might therefore have escaped detection in some species (compare Figure 3).
Figure 2. Conserved editing and 5′ length variation of miR-411. (A) As a result of A-to-I editing (here represented as A-to-G) and alternative 5′ cleavage, the miR-411 precursor gives rise to four variants with distinct seed sequences. The short, unedited variant is the annotated form. (B) Venn diagram of the predicted targets for each miR-411 variant. Targets were predicted with TargetScan release 6.0  and were required to be present in at least 10 species, including human, macaque and mouse. (C) Relative abundance of the four miR-411 variants in (from top to bottom) human, macaque and mouse, when considering reads from all five tissues together.
Figure 3. Frequency of miRNA editing across tissues and species. (A) Estimated miRNA frequencies in brain (B), cerebellum (C), heart (H), kidney (K) and testis (T). From left to right, the animal silhouettes represent human, macaque, mouse, opossum, platypus and chicken. The miRNA identifiers are given in the left-hand column and are followed by the position of the edited site within the mature or star miRNA. The color of each circle corresponds to the proportion of edited reads, while the size of the circle corresponds to the total number of reads for the miRNA in question. Note that these data are not normalized and that expression levels therefore should not be compared across samples. Gray shading indicates the absence of an annotated miRNA ortholog in that particular species, based on the information in Table S1 in Additional file 2. The data used to generate this figure are provided in Table S2 in Additional file 2. (B) Comparison of editing frequencies in neural (brain and cerebellum) versus non-neural (heart, kidney and testis) tissues in human, macaque and mouse, for miRNAs with at least 10 reads in all relevant samples. The median frequency is indicated by a blue line. (C) Comparison of editing frequencies in human and mouse samples for the same miRNAs as in (B). The median frequency is indicated by a blue line. (D) Hierarchical clustering of the same miRNAs as in (B), based on editing frequencies in five tissues in human (hsa), macaque (mml) and mouse (mmu). Orthologous miRNAs have been given the same color. The clustering was performed using the R function hclust and Ward’s method.
Figure 4. Changes in miRNA editing frequencies during primate postnatal development and aging. (A) Spearman correlation coefficients for miRNA editing frequencies and age in human and macaque (see main text for details). The respective correlations for ADAR and ADARB1 mRNA expression levels are indicated as squares. Cases where the correlation coefficients were significant in both species are highlighted in red. (B) Editing frequencies for the six significant miRNAs (see (A)) in samples from different ages, with younger individuals on the left and older on the right. (C) Distribution of correlation coefficients for age and expression levels, calculated for mRNAs predicted to be targeted either by edited or unedited miRNAs. If expression is independent of age, the expected correlation coefficient is 0, as indicated by the dotted line. Statistical significance is indicated by double (0.001 < P < 0.01) or triple (P < 0.001) asterisks.
Figure 5. Comparison of editing frequencies in samples from cancerous and healthy tissues. (A) Each point represents the estimated editing frequency of a single miRNA in a matched sample (cancer and control) from the same patient. Cases where a significant difference in editing was detected between the two conditions (χ2-test with Benjamini-Hochberg correction) are highlighted in red. (B) Summary of the significant cases in (A). Sample identifiers consist of a letter giving the cancer type and a number that refers to the patient identifier in the original study. Blue bars represent significant downregulation of miRNA editing in cancer samples, with the top of the bar corresponding to the editing frequency in the control sample and the bottom of the bar corresponding to the editing frequency in the cancer sample. Red bars represent significant editing upregulation, with the bottom of the bar corresponding to the editing frequency in the control sample and the top of the bar corresponding to the editing frequency in the cancer sample.
Systematic identification of edited microRNAs in the human brain.