Identification of new microRNA-regulated genes by conserved targeting in plant species

Abstract

MicroRNAs (miRNAs) are major regulators of gene expression in multicellular organisms. They recognize their targets by sequence complementarity and guide them to cleavage or translational arrest. It is generally accepted that plant miRNAs have extensive complementarity to their targets and their prediction usually relies on the use of empirical parameters deduced from known miRNA-target interactions. Here, we developed a strategy to identify miRNA targets which is mainly based on the conservation of the potential regulation in different species. We applied the approach to expressed sequence tags datasets from angiosperms. Using this strategy, we predicted many new interactions and experimentally validated previously unknown miRNA targets in Arabidopsis thaliana. Newly identified targets that are broadly conserved include auxin regulators, transcription factors and transporters. Some of them might participate in the same pathways as the targets known before, suggesting that some miRNAs might control different aspects of a biological process. Furthermore, this approach can be used to identify targets present in a specific group of species, and, as a proof of principle, we analyzed Solanaceae-specific targets. The presented strategy can be used alone or in combination with other approaches to find miRNA targets in plants.

Figure 1.

Scheme of the strategy to identify new miRNA targets. The number of detected target genes is indicated for each step of the analysis. After applying the conservation analysis, all genes with the same hit in the Arabidopsis proteome were considered as one target. Note that different genes with the same ID tag give only one hit, so that the total numbers of hits are reduced by this filter. Green squares refer to the target search using empirical filters: bins 5 and 6 include target genes selected by both evolutionary and empirical filters, while bins 2 and 3 have potential targets selected only by evolutionary filters.

Figure 2.

Conservation of potential miRNA targets in different species. The number of targets conserved in different species is indicated for the different miRNAs: all miRNAs (A); miR396 (B), miR408 (C), miR398 (D), miR162 (E) and miR158 (F). The ochre dots represent the targets of the miRNAs using the conservation filter; the light yellow dots show the targets for the randomized miRNAs using the conservation filter. The dark blue squares represent the targets of the miRNAs after applying empirical and evolution filters, while the light blue squares are the targets for the randomized miRNAs under the same conditions. The insets show the specificity, defined as the ratio between the number of targets for the miRNAs and their randomized sequences (ochre dots refer to the targets filtered by their presence in different number of species, while the blue square represents the targets filter by empirical parameters and number of species).

Figure 3.

Selection of miRNA targets by sequence conservation. (A) Relationship between the MFE and the number of species where each target was detected. The MFE represents the average of all cognate target sites. A regression line is indicated. (B) Sensitivity of the approach. The sensitivity was evaluated in two ways, one analyzing the presence of validated targets in Arabidopsis thaliana (light green, described in Supplementary Table S3); and alternatively, it was assayed by the presence of at least one target of each gene family regulated by miRNAs (dark green). (C) Classification of the potential targets present in at least four species.

Figure 4.

Newly validated miRNA targets in Arabidopsis thaliana. The alignments between the miRNAs and their newly identified targets are depicted on the left. The sequence conservation of the miRNA target site in selected species is shown on the right. The figure shows the interaction of miR408 with PAA2 (A); miR408 with PAC1 (B); miR396 with MMG4.7 (C); miR396 with FLU (D); miR159 with NOZZLE (E). The arrows point the position of cleavage sites as determined by 5′ RACE-PCR and the numbers indicate the cloning frequency of each fragment (21).

Figure 5.

Identification of a new target by relaxation of the interaction parameters while increasing the conservation parameter. (A) Scheme showing the strategy to identify the miRNA targets. (B) Conservation of the target site in different species. The arrow indicates a position of a G-C or G-U interaction with the miRNA depending on the species. (C) Alignment of Arabidopsis IAR3 and miR167. The position that contains a G-U wobble is indicated. The arrows show the position of cleavage sites as determined by 5′ RACE-PCR, and the numbers indicate the cloning frequency of each fragment (21).

Figure 6.

Identification of Solanaceae-specific miRNA targets. (A) Prediction of miRNA targets in five Solanaceae species. The number of targets for all conserved miRNAs is indicated after the application of several filters. Targets obtained by the randomized sequences are also indicated. (B) Scheme showing the strategy to identify miRNA targets specific of Solanaceae species. (C) Conservation of the miR398 target site in MT2A sequences from Solanaceae species. (D) Scheme showing the miR398 binding site in tobacco MT2A and MT2B. (E) Transcript levels of CSD2, MT2A and MT2B in wild-type and transgenic tobacco plants (cv. Petit havana) overexpressing miR398. The data shown are mean ± s.e.m. of three biological replicates.