The expansion of the metazoan microRNA repertoire

Abstract

Background: MicroRNAs have been identified as crucial regulators in both animals and plants. Here we report on a comprehensive comparative study of all known miRNA families in animals. We expand the MicroRNA Registry 6.0 by more than 1000 new homologs of miRNA precursors whose expression has been verified in at least one species. Using this uniform data basis we analyze their evolutionary history in terms of individual gene phylogenies and in terms of preservation of genomic nearness across species. This allows us to reliably identify microRNA clusters that are derived from a common transcript.

Results: We identify three episodes of microRNA innovation that correspond to major developmental innovations: A class of about 20 miRNAs is common to protostomes and deuterostomes and might be related to the advent of bilaterians. A second large wave of innovations maps to the branch leading to the vertebrates. The third significant outburst of miRNA innovation coincides with placental (eutherian) mammals. In addition, we observe the expected expansion of the microRNA inventory due to genome duplications in early vertebrates and in an ancestral teleost. The non-local duplications in the vertebrate ancestor are predated by local (tandem) duplications leading to the formation of about a dozen ancient microRNA clusters.

Conclusion: Our results suggest that microRNA innovation is an ongoing process. Major expansions of the metazoan miRNA repertoire coincide with the advent of bilaterians, vertebrates, and (placental) mammals.

Figure 1

Innovations of microRNAs, tandem duplications, and non-local duplications of microRNA genes are unevenly distributed in metazoan phylogeny. Indeed, non-local duplications occur almost exclusively in the ancestral vertebrate and teleosts, resp., in accordance with the 2R/3R model. Species for which large experimental screens for microRNAs have been performed are indicated by a larger font. The phylogenetic tree is based on a recent multi-gene analysis of the major bilaterian groups [69], and the phylogeny of holometabolous insects [70].

Figure 2

(a) Phylogenetic network of mir-1 sequences. Despite the short sequences, the major clades are well separated in this phylogenetic network: there are two vertebrate groups, mir-1-1 and mir-I-2, both of which show a tetrapod and a teleost branch; arthropoda and nematoda are also clearly separated; only the basal deuterostomes do not fit very well due to their diverged sequences. (b) Phylogenetic network of mir-30 sequences, which occur in three clusters each consisting of two miRNAs genes (see inset). A tandem duplication of the ancestral mir-30 sequence gave rise to a single cluster which was duplicated subsequently. Not all details of the duplication history can be resolved due to the short sequence length. It is clear, however, that the duplication events pre-dated the last common ancestor of tetrapoda and teleosts. It is plausible to associate these cluster duplications with the genome duplications at the origin of the vertebrate lineage. Networks were reconstructed using the neighbor net method.

Figure 3

Examples of microRNA gene duplication histories. (a) Gene tree and most plausible reconstructed history of the mir9 cluster. The fourth member of the cluster, mir-306, evolves rapidly in flies. Its homology with mir-9/mir-79 is likely but this hairpin might also have evolved de novo. (b) The two most plausible reconstructions for the history of the mir-23 cluster. Scenario (1) postulates four paralogs in the ancestral vertebrate, where, presumably after the first duplication, one lineage either lost or gained mir-27 in the middle position of the cluster. Subsequently, in this scenario one copy of the three-membered cluster was lost in actinopterygians, while the two-membered clusters were lost in tetrapoda. Scenario (2) postulates three paralogs in the ancestral vertebrate and the independent loss of the mir-27 in two distinct clusters in the teleosts. (c) Duplication history of the mir-130 cluster reconstructed from genomic position information and the gene tree.

Figure 4

Clustalw multiple sequence alignment of mir-421 homologs on the mammalian X chromosome. Additional features (top down): mfe: minimum free energy structure calculated using RNAfold -d2 -noLP, part. func: partition function fold, L2/LINE: direction and position of L2 elements relative to mir-421, mat miRNA: position of mature miRNA, conservat.: conserved positions in sequence alignment.

Figure 5

RNA secondary structures of human (a) and zebrafish (b) mir-220 sequences. Calculations were performed using RNAfold -p -d2 -noLP.

Figure 6

Some microRNA families, such as the mir-10 and mir-100 (left), exhibit very similar mature miRNA sequences, while their precursor sequences show little sequence similarity. Right: A table of alignment z-score for both mature and precursor sequences summarizes the four most likely candidates for distance homologies. While the mir-8/mir-429 pair is most likely a true homolog, the other three pairs are unconvincing, see text.

Figure 7

(a) All microRNAs in the mir-134 cluster appear to have arisen from a common ancestral sequence. The individual paralog groups have diverged rapidly in the ancestor of extant eutherian. Surprisingly, there is very little sequence variation between human and rodents in each of the paralog groups. The six families of alignable microRNAs are indicated in color. (b) WPGMA dendrogram derived from pairwise z-scores of the members of the mir-35 cluster. The analysis of the mature sequences demonstrates that the members of the cluster probably have arisen by means of tandem duplications.