The evolution of microRNA-regulation in duplicated genes facilitates expression divergence

Yonatan Bilu, Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel.

Abstract Background: The evolution of microRNA regulation in metazoans is a mysterious process: MicroRNA sequences are highly conserved among distal organisms, but on the other hand, there is no evident conservation of their targets.
Results: We study this extensive rewiring of the microRNA regulatory network by analyzing the evolutionary trajectories of duplicated genes in D. melanogatser. We find that in general microRNA-targeted genes tend to avoid gene duplication. However, in cases where gene duplication is evident, we find that the gene that displays high divergence from the ancestral gene at the sequence level is also likely to be associated in an opposing manner with the microRNA regulatory system – if the ancestral gene is a miRNA target then the divergent gene tends not to be, and vice versa.
Conclusions: This suggests that miRNAs not only have a role in conferring expression robustness, as was suggested by previous works, but are also an accessible tool in evolving expression divergence.

Background MicroRNAs (miRNA) are short RNA molecules that inhibit protein synthesis by targeting mRNA transcripts [1]. Intriguingly, while miRNAs typically exhibit very high sequence conservation even among evolutionarily distant species, their mRNA targets are specific to each species, with as little overlap as can be expected by chance [2-5]. This portrays the evolution of regulation by miRNAs as a mysterious process, in which the regulatory elements are kept under strong stabilizing selection, while the elements which they regulate are free to diverge.

The lack of target conservation may indicate a high turnover of cis-elements in the 3’ UTR of mRNA transcripts, similar to what has been suggested for transcription factor binding sites (TFBSs) in gene promoter sequences [6-12]. Several works have argued that the latter may, in fact, have little effect on phenotype, as changes can be compensatory, and even when they are not, mRNA expression patterns may often change by way of neutral drift [13, 14]. Could the same be true for miRNA regulation?

Notably, evolution of miRNA regulation is reminiscent, yet ultimately quite different, from regulation mediated by transcription-factors [15]. First, while transcription factors are more conserved than other protein coding genes, they are not as highly conserved as miRNAs. Second, while conservation of TFBSs is lower then might be expected [16-18], it is still relatively higher than for miRNA binding sites, especially among highly conserved transcription factors. Third, transcription-factor binding sites in eukaryotes tend to be short and fuzzy [9, 19]. Binding of miRNA is also thought to be facilitated by short (7bp) sequences [20-22], but less tolerant of fuzziness, perhaps due to a smaller amount of structural cues. Finally, while fitness-neutral changes are possible for both types of regulation, changes in transcription regulation can be more readily compensated for at intermediate levels, which are not relevant for post- transcriptional regulation.

The basic dogma for genetic diversity is that genes undergo duplication, and then one of the copies is free to evolve and explore new functional roles [23-25]. While this view may be simplistic, it highlights the study of gene duplicates as a key tool in understanding evolutionary processes. Indeed, such studies are the basis for identification of whole genome duplication events [23], and cases of subfunctionalization and neofunctionalization [26, 27].

In this work we study the evolution of miRNA regulation as it pertains to gene duplicates in Drosophila melanogaster. We find that miRNA regulation is relatively rare among gene duplicates, possibly due to an indirect selective bias against duplication of miRNA targeted genes. Nonetheless, close examination of miRNA targeting in A. gambiae and D. melanogaster suggests miRNA regulation has an important role in facilitating the exploration of novel functionality. This “exploratory” role may account for the lack of conservation in miRNA targets, and can be seen as complementary to the suggested role of miRNAs in conferring expression robustness [28-30].

Results

miRNA target conservation and 3’ UTR length As previously reported, miRNA target conservation is close to what may be expected at random [15]. Specifically, we compared the targets predicted by miRanda [31] for 14 miRNAs conserved between H. sapiens and D. melanogaster. For each miRNA, we asked how many of the D. melanogaster orthologs of its H. sapiens targets are targeted by it in D. melanogaster (Figure 1). For 9 out of the 14 this number is almost exactly what is expected at random, for 3 it is higher, and for 2 it is, in fact,

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