Argonaute CLIP-Seq reveals miRNA targetome diversity across tissue types

Abstract

To date, analyses of individual targets have provided evidence of a miRNA targetome that extends beyond the boundaries of messenger RNAs (mRNAs) and can involve non-Watson-Crick base pairing in the miRNA seed region. Here we report our findings from analyzing 34 Argonaute HITS-CLIP datasets from several human and mouse cell types. Investigation of the architectural (i.e. bulge vs. contiguous pairs) and sequence (Watson-Crick vs. G:U pairs) preferences for human and mouse miRNAs revealed that many heteroduplexes are "non-canonical" i.e. their seed region comprises G:U and bulge combinations. The genomic distribution of miRNA targets differed distinctly across cell types but remained congruent across biological replicates of the same cell type. For some cell types intergenic and intronic targets were more frequent whereas in other cell types mRNA targets prevailed. The findings suggest an expanded model of miRNA targeting that is more frequent than the standard model currently in use. Lastly, our analyses of data from different cell types and laboratories revealed consistent Ago-loaded miRNA profiles across replicates whereas, unexpectedly, the Ago-loaded targets exhibited a much more dynamic behavior across biological replicates.

Figure 1. Conceptual workflow of CLIPSim-MC.

This schematic depicts the generation of possible expanded-model seed region formations and a CLIP-supported MRE-motif representing a single bulge on the MRE side of the resulting heteroduplex for mouse miR-124-3p.

Figure 2. Examining whether the top expressed miRNAs and MRE clusters recur in the biological replicates of each study.

(a) Spearman correlation across biological replicates for the top-abundant miRNAs. Top row: mouse brain, mouse ESC, and human HEK293. Bottom row: 12 wild type (WT) and 12 miR-155 knockout (155 KO) CD4+ T-cells. (b) Spearman correlation across biological replicates for the statistically significant MRE clusters. Top and bottom rows are as in panel (a) above. (c) Number of unique genomic positions that are captured by all MRE clusters and are common to at least n of the biological replicates available for the tissue or cell type at hand (the value of n is shown on the X-axis). This effectively gauges the degree of recurrence of a specific miRNA target at a specific genomic position (represented by the MRE cluster) across the available biological replicates. (d) In this panel we repeat the calculations of panel (c) considering only the statistically significant MRE clusters in each biological replicate. Note: even though the hTERT-HPNE/MIA PaCa-2 curve does not correspond to biological replicates but to two distinct cell types from the same tissue (pancreas) we include it in panels (c) and (d) for comparison purposes.

Figure 3. Distribution of MRE clusters.

Distribution of statistically significant MRE clusters (p-value ≤ 0.05) across intergenic, intronic and exonic space is shown separately for each sample.

Figure 4. Distribution of paired heteroduplexes.

Panels (a) through (f) show the number of distinct miRNAs associated with a given MRE locus. Data points represent the average over all the replicates of the corresponding sample. Error bars represent the standard deviation across the replicates. The MRE motifs of all considered heteroduplexes have an FDR ≤ 0.5. For ~70% of all miRNA:MRE heteroduplexes we can unambiguously identify a single miRNA for a given MRE.

Figure 5. Distribution of the specific sequence/architecture choices for the seed region among the derived heteroduplexes.

(a) The distribution as seen from the standpoint of miRNAs and separately for each analyzed sample. E.g, according to the shown results, more than 50% of the endogenous miRNAs in MIA PaCa-2 form heteroduplexes with a miRNA-side bulge and at least one wobble in the seed region. (b) The distribution as seen from the standpoint of MREs and separately for each analyzed sample. E.g., according to the shown results, more than 35% of the MIA PaCa-2 MRE's participating in heteroduplexes have a miRNA-side bulge and at least one wobble in the seed region.

Figure 6. Distribution of the number of endogenous miRNAs that are associated with a given number of distinct targets.

(a) Human data. (b) Mouse data. The secondary Y-axis shows the cumulative distribution of the expressed miRNAs that are associated with a given number of targets. Only targets i.e. MREs that are associated with a single miRNA were considered in this calculation.