Adaptive evolution of testis-specific, recently evolved, clustered miRNAs in Drosophila

The propensity of animal miRNAs to regulate targets bearing modest complementarity, most notably via pairing with miRNA positions approximately 2-8 (the "seed"), is believed to drive major aspects of miRNA evolution. First, minimal targeting requirements have allowed most conserved miRNAs to acquire large target cohorts, thus imposing strong selection on miRNAs to maintain their seed sequences. Second, the modest pairing needed for repression suggests that evolutionarily nascent miRNAs may generally induce net detrimental, rather than beneficial, regulatory effects. Hence, levels and activities of newly emerged miRNAs are expected to be limited to preserve the status quo of gene expression. In this study, we unexpectedly show that Drosophila testes specifically express a substantial miRNA population that contravenes these tenets. We find that multiple genomic clusters of testis-restricted miRNAs harbor recently evolved miRNAs, whose experimentally verified orthologs exhibit divergent sequences, even within seed regions. Moreover, this class of miRNAs exhibits higher expression and greater phenotypic capacities in transgenic misexpression assays than do non-testis-restricted miRNAs of similar evolutionary age. These observations suggest that these testis-restricted miRNAs may be evolving adaptively, and several methods of evolutionary analysis provide strong support for this notion. Consistent with this, proof-of-principle tests show that orthologous miRNAs with divergent seeds can distinguish target sensors in a species-cognate manner. Finally, we observe that testis-restricted miRNA clusters exhibit extraordinary dynamics of miRNA gene flux in other Drosophila species. Altogether, our findings reveal a surprising tissue-directed influence of miRNA evolution, involving a distinct mode of miRNA function connected to adaptive gene regulation in the testis

Detection and Verification of Mammalian Mirtrons by Northern Blotting

microRNAs (miRNAs) have vital roles in regulating gene expression—contributing to major diseases like cancer and heart disease. Over the last decade, thousands of miRNAs have been discovered through high throughput sequencing-based annotation. Different classes have been described, as well as a great dynamic range of expression levels. While sequencing approaches provide insight into biogenesis and allow confident identification, there is a need for additional methods for validation and characterization. Northern blotting was one of the first techniques used for studying miRNAs, and remains one of the most valuable as it avoids enzymatic manipulation of miRNA transcripts. Blotting can also provide insight into biogenesis by revealing RNA processing intermediates. Compared to sequencing, however, northern blotting is a relatively insensitive technology. This creates a challenge for detecting low expressed miRNAs, particularly those produced by inefficient, non-canonical pathways. In this chapter, we describe a strategy to study such miRNAs by northern blotting that involves ectopic expression of both miRNAs and miRNA-binding Argonaute (Ago) proteins. Through use of epitope tags, this strategy also provides a convenient method for verification of small RNA competency to be loaded into regulatory complexes

Uridylation and adenylation of RNAs

The posttranscriptional addition of nontemplated nucleotides to the 3′ ends of RNA molecules can have a significant impact on their stability and biological function. It has been recently discovered that nontemplated addition of uridine or adenosine to the 3′ ends of RNAs occurs in different organisms ranging from algae to humans, and on different kinds of RNAs, such as histone mRNAs, mRNA fragments, U6 snRNA, mature small RNAs and their precursors etc. These modifications may lead to different outcomes, such as increasing RNA decay, promoting or inhibiting RNA processing, or changing RNA activity. Growing pieces of evidence have revealed that such modifications can be RNA sequence-specific and subjected to temporal or spatial regulation in development. RNA tailing and its outcomes have been associated with human diseases such as cancer. Here, we review recent developments in RNA uridylation and adenylation and discuss the future prospects in this research area

Importance of miRNA stability and alternative primary miRNA isoforms in gene regulation during Drosophila development

10.7554/eLife.38389eLife7e3838

Evolution of mir-92a underlies natural morphological variation in drosophila melanogaster

Identifying the genetic mechanisms underlying phenotypic change is essential to understanding how gene regulatory networks and ultimately the genotype-to-phenotype map evolve. It is recognized that microRNAs (miRNAs) have the potential to facilitate evolutionary change [1–3]; however, there are no known examples of natural morphological variation caused by evolutionary changes in miRNA expression. Therefore, the contribution of miRNAs to evolutionary change remains unknown [1, 4]. Drosophila melanogaster subgroup species display a portion of trichome-free cuticle on the femur of the second leg called the “naked valley.” It was previously shown that Ultrabithorax (Ubx) is involved in naked valley variation between D. melanogaster and D. simulans [5, 6]. However, naked valley size also varies among populations of D. melanogaster, ranging from 1,000 up to 30,000 μm(2). We investigated the genetic basis of intraspecific differences in the naked valley in D. melanogaster and found that neither Ubx nor shavenbaby (svb) [7, 8] contributes to this morphological difference. Instead, we show that changes in mir-92a expression underlie the evolution of naked valley size in D. melanogaster through repression of shavenoid (sha) [9]. Therefore, our results reveal a novel mechanism for morphological evolution and suggest that modulation of the expression of miRNAs potentially plays a prominent role in generating organismal diversity

A genome-wide transgenic resource for conditional expression of Drosophila microRNAs.

microRNAs (miRNAs) are endogenous short RNAs that mediate vast networks of post-transcriptional gene regulation. Although computational searches and experimental profiling provide evidence for hundreds of functional targets for individual miRNAs, such data rarely provide clear insight into the phenotypic consequences of manipulating miRNAs in vivo. We describe a genome-wide collection of 165 Drosophila miRNA transgenes and find that a majority induced specific developmental defects, including phenocopies of mutants in myriad cell-signaling and patterning genes. Such connections allowed us to validate several likely targets for miRNA-induced phenotypes. Importantly, few of these phenotypes could be predicted from computationally predicted target lists, thus highlighting the value of whole-animal readouts of miRNA activities. Finally, we provide an example of the relevance of these data to miRNA loss-of-function conditions. Whereas misexpression of several K box miRNAs inhibited Notch pathway activity, reciprocal genetic interaction tests with miRNA sponges demonstrated endogenous roles of the K box miRNA family in restricting Notch signaling. In summary, we provide extensive evidence that misexpression of individual miRNAs often induces specific mutant phenotypes that can guide their functional study. By extension, these data suggest that the deregulation of individual miRNAs in other animals may frequently yield relatively specific phenotypes during disease conditions

eworoRL514BC_QTL

Phenotype and genotype data for QTL mapping between strains e, wo, ro and RAL514

Data from: Evolution of mir-92a underlies natural morphological variation in Drosophila melanogaster

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