Canalization of Phenotypes-When the Transcriptome is Constantly but Weakly Perturbed.

Recent studies have increasingly pointed to microRNAs (miRNAs) as the agent of gene regulatory network (GRN) stabilization as well as developmental canalization against constant but small environmental perturbations. To analyze mild perturbations, we construct a Dicer-1 knockdown line (dcr-1 KD) in Drosophila that modestly reduces all miRNAs by, on average, ∼20%. The defining characteristic of stabilizers is that, when their capacity is compromised, GRNs do not change their short-term behaviors. Indeed, even with such broad reductions across all miRNAs, the changes in the transcriptome are very modest during development in stable environment. By comparison, broad knockdowns of other regulatory genes (esp. transcription factors) by the same method should lead to drastic changes in the GRNs. The consequence of destabilization may thus be in long-term development as postulated by the theory of canalization. Flies with modest miRNA reductions may gradually deviate from the developmental norm, resulting in late-stage failures such as shortened longevity. In the optimal culture condition, the survival to adulthood is indeed normal in the dcr-1 KD line but, importantly, adult longevity is reduced by ∼90%. When flies are stressed by high temperature, dcr-1 KD induces lethality earlier in late pupation and, as the perturbations are shifted earlier, the affected stages are shifted correspondingly. Hence, in late stages of development with deviations piling up, GRN would be increasingly in need of stabilization. In conclusion, miRNAs appear to be a solution to weak but constant environmental perturbations.

Spatiotemporal Regulation of a Single Adaptively Evolving Trans-Regulatory Element Contributes to Spermatogenetic Expression Divergence in Drosophila.

Due to extensive pleiotropy, trans-acting elements are often thought to be evolutionarily constrained. While the impact of trans-acting elements on gene expression evolution has been extensively studied, relatively little is understood about the contribution of a single trans regulator to interspecific expression and phenotypic divergence. Here, we disentangle the effects of genomic context and miR-983, an adaptively evolving young microRNA, on expression divergence between Drosophila melanogaster and D. simulans. We show miR-983 effects promote interspecific expression divergence in testis despite its antagonism with the often-predominant context effects. Single-cyst RNA-seq reveals that distinct sets of genes gain and lose miR-983 influence under disruptive or diversifying selection at different stages of spermatogenesis, potentially helping minimize antagonistic pleiotropy. At the round spermatid stage, the effects of miR-983 are weak and distributed, coincident with the transcriptome undergoing drastic expression changes. Knocking out miR-983 causes reduced sperm length with increased within-individual variation in D. melanogaster but not in D. simulans, and the D. melanogaster knockout also exhibits compromised sperm defense ability. Our results provide empirical evidence for the resolution of antagonistic pleiotropy and also have broad implications for the function and evolution of new trans regulators.

Run or Die in the Evolution of New MicroRNAs-Testing the Red Queen Hypothesis on De Novo New Genes.

The Red Queen hypothesis depicts evolution as the continual struggle to adapt. According to this hypothesis, new genes, especially those originating from nongenic sequences (i.e., de novo genes), are eliminated unless they evolve continually in adaptation to a changing environment. Here, we analyze two Drosophila de novo miRNAs that are expressed in a testis-specific manner with very high rates of evolution in their DNA sequence. We knocked out these miRNAs in two sibling species and investigated their contributions to different fitness components. We observed that the fitness contributions of miR-975 in Drosophila simulans seem positive, in contrast to its neutral contributions in D. melanogaster, whereas miR-983 appears to have negative contributions in both species, as the fitness of the knockout mutant increases. As predicted by the Red Queen hypothesis, the fitness difference of these de novo miRNAs indicates their different fates.

Dynamic global analysis of transcription reveals the role of miRNAs in synergistic stabilization of gene expression.

Buffering exogenous perturbation is crucial to maintain transcriptional homeostasis during development. While miRNAs have been speculated to play a role in stability maintenance, previous studies seeking to check this conjecture focused on measurements of transcript levels at steady state or involved individual miRNA targets. We measured whole-genome expression dynamics by introducing a transient perturbation and establishing a perturbation and recovery system in Drosophila larvae. We inhibited all transcription and assayed transcriptomes at several time points during recovery from inhibition. We performed these experiments in the wild type and miRNA-deficient genetic backgrounds. Consistent with theories about miRNAs' function in stabilizing the transcriptome, we find that attenuating miRNA expression leads to weak impairment in degradation of targets but strong destabilization of target genes when transcription is re-activated. We further fitted a model that captures the essential aspects of transcription dynamics in our experiments and found that the miRNA target transcripts uniformly overshoot the original steady state as they recover from a general inhibition of transcription if global miRNA levels are reduced. Collectively, our results provide experimental evidence for the idea that miRNAs act cumulatively to stabilize the transcriptional regulatory network. We therefore found a promising approach to assess the effect of these molecules on transcription dynamics.

Death of new microRNA genes in Drosophila via gradual loss of fitness advantages.

The prevalence of de novo coding genes is controversial due to length and coding constraints. Noncoding genes, especially small ones, are freer to evolve de novo by comparison. The best examples are microRNAs (miRNAs), a large class of regulatory molecules ∼22 nt in length. Here, we study six de novo miRNAs in Drosophila, which, like most new genes, are testis-specific. We ask how and why de novo genes die because gene death must be sufficiently frequent to balance the many new births. By knocking out each miRNA gene, we analyzed their contributions to the nine components of male fitness (sperm production, length, and competitiveness, among others). To our surprise, the knockout mutants often perform better than the wild type in some components, and slightly worse in others. When two of the younger miRNAs are assayed in long-term laboratory populations, their total fitness contributions are found to be essentially zero. These results collectively suggest that adaptive de novo genes die regularly, not due to the loss of functionality, but due to the canceling out of positive and negative fitness effects, which may be characterized as "quasi-neutrality." Since de novo genes often emerge adaptively and become lost later, they reveal ongoing period-specific adaptations, reminiscent of the "Red-Queen" metaphor for long-term evolution.

On the possibility of death of new genes - evidence from the deletion of de novo microRNAs.

BACKGROUND: New genes are constantly formed, sometimes from non-genic sequences, creating what is referred to as de novo genes. Since the total number of genes remains relatively steady, gene deaths likely balance out new births. In metazoan genomes, microRNAs (miRs) genes, small and non-coding, account for the bulk of functional de novo genes and are particularly suited to the investigation of gene death. RESULTS: In this study, we discover a Drosophila-specific de novo miRNA (mir-977) that may be facing impending death. Strikingly, after this testis-specific gene is deleted from D. melanogaster, most components of male fitness increase, rather than decrease as had been expected. These components include male viability, fertility and males' ability to repress female re-mating. Given that mir-977 has a negative fitness effect in D. melanogaster, this de novo gene with an adaptive history for over 60 Myrs may be facing elimination. In some other species where mir-977 is not found, gene death may have already happened. CONCLUSION: The surprising result suggests that de novo genes, constantly rising and falling during evolution, may often be transiently adaptive and then purged from the genome.