Computational prediction and experimental validation of evolutionarily conserved microRNA target genes in bilaterian animals

Abstract

Background: In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution.

Results: Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells.

Conclusions: The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions.

Figure 1

Computational extraction of conserved miRNA/target gene pairs among bilaterian animals. Evolutionarily conserved miRNAs were extracted from the five model species (H. sapiens, M. musculus, G. gallus, D. melanogaster, and C. elegans). For each miRNA, potential target genes were predicted using the following criteria: optimal free-energy threshold and complete matching of nucleotide sequences between the seed sequence of miRNA/mRNA duplexes (step 1), binding pattern of the 3'-UTR of miRNA/mRNA duplexes (step 2), and orthologous gene information (step 3).

Figure 2

Parameters used for the prediction of miRNA/mRNA pairs and their coverage. To optimize the binding parameters of miRNA/mRNA duplexes, we determined the coverage of four binding parameters (mismatch of mRNA within the whole miRNA sequence (A), mismatch of miRNA within the whole miRNA sequence (B), G-U wobble pairs within the whole miRNA sequence (C), and G-U wobble pairs within the seed sequence (D)). Calculation of the coverage was performed using 112 experimentally verified miRNA/mRNA pairs (orange line) and 153,387 predicted miRNA/mRNA pairs (blue line). The yellow bar indicates differences in coverage between experimentally verified and computationally predicted miRNA/mRNA pairs. Four or five points chosen from the highest yellow bar were used as the range of each of the four binding parameters (blue squares) used in this study. We determined parameter space using the binding patterns of the miRNA/mRNA pairs based on four features (E). Five hundred parameter combinations were plotted on a 2D graph using "ratio of experimentally verified miRNA/mRNA" on the Y-axis and "Enrichment" on the X-axis. Black circles (57.3% of the coverage and 3.2-fold of the Enrichment) indicate the point that corresponded to optimized parameters for the prediction of final conserved miRNA/target pairs: 12 for the number of mismatches in the mRNA, 10 for the number of mismatches in the miRNA, 4 for the number of G-U wobble pairs within the whole miRNA sequence, and 0 for the number of G-U wobble pairs within the seed sequence (see Methods section).

Figure 3

Two examples of miRNA target sites in orthologous gene transcripts. Potential target sites of miR-1 (blue arrows) in the 3'-UTR sequences of the orthologous CNN3 (A) and LARP4 (B) transcripts are shown. Predicted duplexes formed by the 3'-UTR sequences (top) and miRNAs (bottom) are shown in dotted boxes for each potential target site. The green bar on the H. sapiens 3'-UTR sequence indicates a DNA region used for the construction of the reporter plasmid pLuc-CNN3 (Figure 4A). See Additional file 3 for other candidates. (*) The length of the CNN3 3'-UTR is currently registered as a little shorter than that indicated (527 nt in size) and contains the miR-1 binding site (Ensembl release 53).

Figure 4

Example of the 3'-UTR reporter plasmid and experimental validation. The 3'-UTR sequences of DLL1, CNN3, LARP4, ELK3, EIF2C4, TAGLN2, and ATP6V1B2 were subcloned into the XhoI/NotI site of the psiCHECK™-2 vector. CNN3 was chosen as representative of the eight candidates listed above (see Methods section). (B) HeLa cells were cotransfected with each combination of 100 ng of reporter plasmid and the indicated amounts of each miRNA (DLL1, 5 pmol of miR-34 (a); CNN3, 60 pmol of miR-1 (b); LARP4, 20 pmol of miR-1 (c); ELK3, 60 pmol of miR-124 (d); EIF2C4, 60 pmol of let-7 (e), TAGLN2, 5 pmol of miR-1 (f), ATP6V1V2, 60 pmol of miR-1 (g), and LARP4, 60 pmol of let-7 (h)). Colours depict each miRNA: miR-34 (red), miR-1 (blue), miR-124 (orange), let-7 (green), and negative control (black). The relative expression of the luciferase gene was measured 24 h after transfection. The normalized luciferase activity of the control vector was set as 1.0. The data represent the average of three experiments and SDs. * P < 0.01.