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. 2012 Dec 14;48(5):760-70.
doi: 10.1016/j.molcel.2012.10.002. Epub 2012 Nov 8.

Transcriptome-wide miR-155 binding map reveals widespread noncanonical microRNA targeting

Gabriel B Loeb  1 , Aly A Khan, David Canner, Joseph B Hiatt, Jay Shendure, Robert B Darnell, Christina S Leslie, Alexander Y Rudensky
Affiliations

Transcriptome-wide miR-155 binding map reveals widespread noncanonical microRNA targeting

Gabriel B Loeb et al. Mol Cell. .

Abstract

MicroRNAs (miRNAs) are essential components of gene regulation, but identification of miRNA targets remains a major challenge. Most target prediction and discovery relies on perfect complementarity of the miRNA seed to the 3' untranslated region (UTR). However, it is unclear to what extent miRNAs target sites without seed matches. Here, we performed a transcriptome-wide identification of the endogenous targets of a single miRNA-miR-155-in a genetically controlled manner. We found that approximately 40% of miR-155-dependent Argonaute binding occurs at sites without perfect seed matches. The majority of these noncanonical sites feature extensive complementarity to the miRNA seed with one mismatch. These noncanonical sites confer regulation of gene expression, albeit less potently than canonical sites. Thus, noncanonical miRNA binding sites are widespread, often contain seed-like motifs, and can regulate gene expression, generating a continuum of targeting and regulation.

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Figures

Figure 1
Figure 1. Differential CLIP-sequencing identifies sites of miR-155 mediated Argonaute binding
(a) CD4+ T cells were isolated from miR-155-sufficient and - deficient mice and RNase treated cell lysates were subjected to Argonaute (AGO) immunoprecipitation. Following stringent purification of complexes, indexed and barcoded libraries were prepared and subjected to Illumina high-throughput sequencing. (b) Examples of AGO binding sites in novel (PD-L1) and established (SHIP1) canonical targets. Grey rectangles indicate miR-155-dependent binding sites (p < .01) called by edge detection method. Reads from the 12 replicates have been stacked; blue shades are miR-155-sufficient (WT) replicates, and yellow shades are mir-155-deficient (155KO) replicates. Black bars indicate the location of the miR-155 seed in the binding site. Coordinates along the x-axis indicate nucleotide position relative to the beginning of the 3′UTR. The y-axis indicates read counts, which are square root transformed following individual library normalization (see Computational Methods). (c) Relative AGO binding in WT and 155KO cells at all 3′UTR binding sites, or 3′UTR binding sites containing a miR-155 seed. (d) Gene expression differences assessed by microarray between WT and 155KO cells of targets predicted by seed, AGO binding sites, or miR-155 dependent AGO binding sites. p-values were calculated by using a one-sided KS test.
Figure 2
Figure 2. Argonaute binding to coding sequence
(a) Proportion of reads from miR-155-sufficient (WT) and miR-155-deficient (155KO) libraries mapping to various regions of the genome. (b) Examples of AGO binding sites in the coding region. Coordinates along the x-axis indicate nucleotide position relative to the beginning of the transcript. (c) Relative AGO binding in WT and 155KO cells at all binding sites, 3′UTR binding sites containing a miR-155 seed, or CDS binding sites containing a miR-155 seed. (d) Gene expression differences assessed by microarray between WT and 155KO cells of targets predicted by seed and AGO binding sites in the coding region. p-values were calculated using a one-sided KS test.
Figure 3
Figure 3. Many miRNA-dependent sites do not contain seed matches
(a) Proportion of sites from all AGO binding sites or from miR-155-dependent sites that do not contain seed matches. (b-c) Examples of non-canonical targets. Dotted black vertical lines indicate the location of a seed-like site in the site. Predicted base-pairing between miR-155 and 3′UTR sequences are indicated below AGO dCLIP read distribution profiles. Positions selected for mutations in luciferase reporter constructs are highlighted in the alignment. (c) Example of a gene with both a non-canonical and a canonical site in the same 3′UTR; numbers along the horizontal axis indicate nucleotides from the start of the 3′UTR.
Figure 4
Figure 4. Characteristics of non-canonical sites
(a) Variations in canonical (top) and non-canonical (bottom) motifs in miR-155-dependent sites. Number in parentheses indicates the number of miR-155-dependent AGO binding sites containing the motif. (b) Cross-linking induced deletions around canonical seed matches and non-canonical seed-like motifs. (c) Enrichment for inexact 6mer and 7mer motifs in non-canonical miR-155-dependent binding sites relative to all binding sites. (d) Enrichment of 3′ complementarity in non-canonical miR-155 binding sites. Enrichment of motifs complementary to miR-155 was examined in a 16-nucleotide regions found 3-18nt 5′ of inexact seed matches in non-canonical sites relative to equally sized regions in Argonaute binding sites lacking miR-155 seed matches. Enrichments were calculated using Fisher’s Exact Test. (e) AU content in a 100nt window centered at seed matches or non-canonical seed-like motifs.
Figure 5
Figure 5. Gene regulation by non-canonical sites
(a) Gene expression differences assessed by microarray for canonical targets and non-canonical targets without seed matches in the 3′UTR in miR-155-sufficient and -deficient T cells. (b) Repression of 3′UTRs with non-canonical target sites by miR-155 assessed in luciferase reporter assays. Percent repression is the reduction in luciferase activity observed upon miR-155 overexpression relative to the luciferase activity observed with control miRNA overexpression. Socs1, a canonical target, the Socs1 miR-155 site mutant and Tgm2, were used as a positive and negative controls, respectively. (c) Repression of luciferase reporter expression for constructs with and without mutations in the predicted non-canonical sites. Mutations are shown in Figure 3b. (d) Gene expression differences from microarray data (Mu et al., 2009) of canonical targets and non-canonical targets that lacked miR-17~92 seed matches anywhere in the 3′UTR in wild type and miR-17~92 cluster deficient transformed B cells. Predicted non-canonical targets contain AGO binding sites with a single mismatch from miR-17~92 seed matches and an exact match to a 3′ motif. In (b) and (c) mean ± SEM is plotted, *, p<0.05; **, p<0.01; ****, p<10-4.
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