Novel regulation and functional interaction of polycistronic miRNAs

The importance of microRNAs in gene expression and disease is well recognized. However, what is less appreciated is that almost half of miRNA genes are organized in polycistronic clusters and are therefore coexpressed. The mir-11∼998 cluster consists of two miRNAs, miR-11 and miR-998. Here, we describe a novel layer of regulation that links the processing and expression of miR-998 to the presence of the mir-11 gene. We show that the presence of miR-11 in the pri-miRNA is required for processing by Drosha, and deletion of mir-11 prevents the expression of miR-998. Replacing mir-11 with an unrelated miRNA rescued miR-998 expression in vivo and in vitro, as did expressing miR-998 from a shorter, more canonical miRNA scaffold. The embedded regulation of miR-998 is functionally important because unchecked miR-998 expression in the absence of miR-11 resulted in pleiotropic developmental defects. This novel regulation of expression of miRNAs within a cluster is not limited to the mir-11∼998 cluster and, thus, likely reflects the more general cis-regulation of expression of individual miRNAs. Collectively, our results uncover a novel layer of regulation within miRNA clusters that tempers the functions of the individual miRNAs. Unlinking their expression has the potential to change the expression of multiple miRNA targets and shift a biological response.     

Transcriptome Analysis of Honeybee (Apis Mellifera) Haploid and Diploid Embryos Reveals Early Zygotic Transcription during Cleavage

In honeybees, the haplodiploid sex determination system promotes a unique embryogenesis process wherein females develop from fertilized eggs and males develop from unfertilized eggs. However, the developmental strategies of honeybees during early embryogenesis are virtually unknown. Similar to most animals, the honeybee oocytes are supplied with proteins and regulatory elements that support early embryogenesis. As the embryo develops, the zygotic genome is activated and zygotic products gradually replace the preloaded maternal material. The analysis of small RNA and mRNA libraries of mature oocytes and embryos originated from fertilized and unfertilized eggs has allowed us to explore the gene expression dynamics in the first steps of development and during the maternal-to-zygotic transition (MZT). We localized a short sequence motif identified as TAGteam motif and hypothesized to play a similar role in honeybees as in fruit flies, which includes the timing of early zygotic expression (MZT), a function sustained by the presence of the zelda ortholog, which is the main regulator of genome activation. Predicted microRNA (miRNA)-target interactions indicated that there were specific regulators of haploid and diploid embryonic development and an overlap of maternal and zygotic gene expression during the early steps of embryogenesis. Although a number of functions are highly conserved during the early steps of honeybee embryogenesis, the results showed that zygotic genome activation occurs earlier in honeybees than in Drosophila based on the presence of three primary miRNAs (pri-miRNAs) (ame-mir-375, ame-mir-34 and ame-mir-263b) during the cleavage stage in haploid and diploid embryonic development.     

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.     

Patterns of molecular evolution of the germ line specification gene oskar suggest that a novel domain may contribute to functional divergence in Drosophila

In several metazoans including flies of the genus Drosophila, germ line specification occurs through the inheritance of maternally deposited cytoplasmic determinants, collectively called germ plasm. The novel insect gene oskar is at the top of the Drosophila germ line specification pathway, and also plays an important role in posterior patterning. A novel N-terminal domain of oskar (the Long Oskar domain) evolved in Drosophilids, but the role of this domain in oskar functional evolution is unknown. Trans-species transgenesis experiments have shown that oskar orthologs from different Drosophila species have functionally diverged, but the underlying selective pressures and molecular changes have not been investigated. As a first step toward understanding how Oskar function could have evolved, we applied molecular evolution analysis to oskar sequences from the completely sequenced genomes of 16 Drosophila species from the Sophophora subgenus, Drosophila virilis and Drosophila immigrans. We show that overall, this gene is subject to purifying selection, but that individual predicted structural and functional domains are subject to heterogeneous selection pressures. Specifically, two domains, the Drosophila-specific Long Osk domain and the region that interacts with the germ plasm protein Lasp, are evolving at a faster rate than other regions of oskar. Further, we provide evidence that positive selection may have acted on specific sites within these two domains on the D. virilis branch. Our domain-based analysis suggests that changes in the Long Osk and Lasp-binding domains are strong candidates for the molecular basis of functional divergence between the Oskar proteins of D. melanogaster and D. virilis. This molecular evolutionary analysis thus represents an important step towards understanding the role of an evolutionarily and developmentally critical gene in germ plasm evolution and assembly.     

The structure and evolution of cis-regulatory regions: the shavenbaby story

In this paper, we provide a historical account of the contribution of a single line of research to our current understanding of the structure of cis-regulatory regions and the genetic basis for morphological evolution. We revisit the experiments that shed light on the evolution of larval cuticular patterns within the genus Drosophila and the evolution and structure of the shavenbaby gene. We describe the experiments that led to the discovery that multiple genetic changes in the cis-regulatory region of shavenbaby caused the loss of dorsal cuticular hairs (quaternary trichomes) in first instar larvae of Drosophila sechellia. We also discuss the experiments that showed that the convergent loss of quaternary trichomes in D. sechellia and Drosophila ezoana was generated by parallel genetic changes in orthologous enhancers of shavenbaby. We discuss the observation that multiple shavenbaby enhancers drive overlapping patterns of expression in the embryo and that these apparently redundant enhancers ensure robust shavenbaby expression and trichome morphogenesis under stressful conditions. All together, these data, collected over 13 years, provide a fundamental case study in the fields of gene regulation and morphological evolution, and highlight the importance of prolonged, detailed studies of single genes.     

Adaptive Evolution and the Birth of CTCF Binding Sites in the Drosophila Genome

Changes in the physical interaction between cis-regulatory DNA sequences and proteins drive the evolution of gene expression. However, it has proven difficult to accurately quantify evolutionary rates of such binding change or to estimate the relative effects of selection and drift in shaping the binding evolution. Here we examine the genome-wide binding of CTCF in four species of Drosophila separated by between ∼2.5 and 25 million years. CTCF is a highly conserved protein known to be associated with insulator sequences in the genomes of human and Drosophila. Although the binding preference for CTCF is highly conserved, we find that CTCF binding itself is highly evolutionarily dynamic and has adaptively evolved. Between species, binding divergence increased linearly with evolutionary distance, and CTCF binding profiles are diverging rapidly at the rate of 2.22% per million years (Myr). At least 89 new CTCF binding sites have originated in the Drosophila melanogaster genome since the most recent common ancestor with Drosophila simulans. Comparing these data to genome sequence data from 37 different strains of Drosophila melanogaster, we detected signatures of selection in both newly gained and evolutionarily conserved binding sites. Newly evolved CTCF binding sites show a significantly stronger signature for positive selection than older sites. Comparative gene expression profiling revealed that expression divergence of genes adjacent to CTCF binding site is significantly associated with the gain and loss of CTCF binding. Further, the birth of new genes is associated with the birth of new CTCF binding sites. Our data indicate that binding of Drosophila CTCF protein has evolved under natural selection, and CTCF binding evolution has shaped both the evolution of gene expression and genome evolution during the birth of new genes.     

Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila

Juvenile hormone (JH) signaling underpins both regulatory and developmental pathways in insects. However, the JH receptor is poorly understood. Methoprene tolerant (Met) and germ cell expressed (gce) have been implicated in JH signaling in Drosophila. We investigated the evolution of Met and gce across 12 Drosophila species and found that these paralogs are conserved across at least 63 million years of dipteran evolution. Distinct patterns of selection found using estimates of dN/dS ratios across Drosophila Met and gce coding sequences, along with their incongruent temporal expression profiles in embryonic Drosophila melanogaster, illustrate avenues through which these genes have diverged within the Diptera. Additionally, we demonstrate that the annotated gene CG15032 is the 5′ terminus of gce.In mosquitoes and beetles, a single Met-like homolog displays structural similarity to both Met and gce, and the intron locations are conserved with those of gce. We found that Tribolium and mosquito Met orthologs are assembled from Met- and gce-specific domains in a modular fashion. Our results suggest that Drosophila Met and gce experienced divergent evolutionary pressures following the duplication of an ancestral gce-like gene found in less derived holometabolous insects.     

Postgastrular zen expression is required to develop distinct amniotic and serosal epithelia in the scuttle fly Megaselia

The amnioserosa is an extraembryonic epithelium that evolved in higher cyclorrhaphan flies from distinct serosal and amniotic epithelia. The underlying genetic mechanism of this evolutionary transition is unknown. Amnioserosa development of Drosophila correlates with novel expression characteristics of the homeobox gene zerknüllt (zen), including a broad zen expression domain in the syncytial blastoderm and the complete absence of postgastrular zen expression. Here we examine the functional significance of these features by altering the activity profile of zen in Megaselia (a lower cyclorrhaphan fly with distinct serosal and amniotic epithelia) and Drosophila, and by examining in Megaselia the function of u-shaped group (ush-group) genes, which in Drosophila maintain the amnioserosa after gastrulation when zen is no longer expressed. In Megaselia, loss of postgastrular zen expression abrogates serosa development but allows amnion development. Ectopic expression of zen in early Megaselia embryos allows serosa formation but perturbs amnion development. Megaselia homologues of u-shaped group genes are not essential for serosa formation but mediate germband retraction and dorsal closure. Finally, ectopic postgastrular zen expression in Drosophila causes an enlargement of amnioserosa cells and interferes with the morphogenetic functions of the amnioserosa. Our results suggest that the origin of the amnioserosa involved the loss of postgastrular zen expression from extraembryonic tissue, that the early broad expression domain of Drosophila zen evolved afterwards, and that the ush-group genes ancestrally played a role in morphogenetic functions of the amnion.     

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 ∼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.