Nicholas S. Sokol

Hormonal activation of let-7-C microRNAs via EcR is required for adult Drosophila melanogaster morphology and function

Steroid hormones and their nuclear receptors drive developmental transitions in diverse organisms, including mammals. In this study, we show that the Drosophila steroid hormone 20-hydroxyecdysone (20E) and its nuclear receptor directly activate transcription of the evolutionarily conserved let-7-complex (let-7-C) locus, which encodes the co-transcribed microRNAs miR-100, let-7 and miR-125. These small RNAs post-transcriptionally regulate the expression of target genes, and are required for the remodeling of the Drosophila neuromusculature during the larval-to-adult transition. Deletion of three 20E responsive elements located in the let-7-C locus results in reduced levels of let-7-C microRNAs, leading to neuromuscular and behavioral defects in adults. Given the evolutionary conservation of let-7-C microRNA sequences and temporal expression profiles, these findings indicate that steroid hormone-coupled control of let-7-C microRNAs is part of an ancestral pathway controlling the transition from larval-to-reproductive animal forms.

MicroRNAs in Drosophila Development

Micro-ribonucleic acids (miRNAs) are small (21-24 nucleotide), endogenously expressed, noncoding RNAs that have emerged as important posttranscriptional regulators of gene expression. MiRNAs have been identified and cloned from diverse eukaryotic organisms where they have been shown to control important physiological and developmental processes such as apoptosis, cell division, and differentiation. A high level of conservation of some miRNAs across phyla further emphasizes their importance as posttranscriptional regulators. Research in a variety of model systems has been instrumental in dissecting the biological functions of miRNAs. In this chapter, we discuss the current literature on the role of miRNAs as developmental regulators in Drosophila.

Small temporal RNAs in animal development.

The lin-4/miR-125 and let-7 microRNAs are at the heart of the heterochronic pathway, which controls temporal cell fate determination during Caenorhabditis elegans development. These small temporal RNAs are clustered along with a third microRNA, miR-100, in the genomes of most animals. Their conserved temporal and neural expression profile suggests a general role in cell fate determination during nervous system differentiation. By triggering consecutive differentiation programs, these microRNAs probably help to determine birth-order dependent temporal identity and thereby contribute to neural stem cell multipotency.

The Role of MicroRNAs in Muscle Development

MicroRNAs play essential roles during animal development, including in developing muscle. Many microRNAs are expressed during muscle development and some, like miR-1 and miR-133, are muscle specific. Muscle microRNAs are integrated into myogenic regulatory networks: their expression is under the transcriptional and posttranscriptional control of myogenic factors, and they in turn have widespread control of muscle gene expression. This review summarizes recent work characterizing the function of microRNAs in muscle biology and specifically focuses on the genetic analysis of muscle microRNAs in a variety of model organisms including worms, flies, zebrafish, and mice.

Neural stem cell-encoded temporal patterning delineates an early window of malignant susceptibility in Drosophila

Pediatric neural tumors are often initiated during early development and can undergo very rapid transformation. However, the molecular basis of this early malignant susceptibility remains unknown. During Drosophila development, neural stem cells (NSCs) divide asymmetrically and generate intermediate progenitors that rapidly differentiate in neurons. Upon gene inactivation, these progeny can dedifferentiate and generate malignant tumors. Here, we find that intermediate progenitors are prone to malignancy only when born during an early window of development while expressing the transcription factor Chinmo, and the mRNA-binding proteins Imp/IGF2BP and Lin-28. These genes compose an oncogenic module that is coopted upon dedifferentiation of early-born intermediate progenitors to drive unlimited tumor growth. In late larvae, temporal transcription factor progression in NSCs silences the module, thereby limiting mitotic potential and terminating the window of malignant susceptibility. Thus, this study identifies the gene regulatory network that confers malignant potential to neural tumors with early developmental origins. DOI: http://dx.doi.org/10.7554/eLife.13463.001

Drosha-independent DGCR8/Pasha pathway regulates neuronal morphogenesis

Significance Understanding the neuronal functions of diverse RNA pathways will lead to treatments of human neurological diseases that are caused by perturbations in RNA metabolism. Two proteins, Drosha and Pasha/DGCR8, play important roles in neurons, where they are responsible for the biogenesis of many microRNAs. Here, we show that Pasha/DGCR8 also promotes the morphogenesis of neurons in developing fruit flies independently of Drosha and therefore of most microRNA production. These studies therefore illuminate a novel function of Pasha that is medically relevant, because loss of the human ortholog of Pasha may contribute to cognitive and behavioral disorders associated with DiGeorge syndrome. Cleavage of microRNAs and mRNAs by Drosha and its cofactor Pasha/DGCR8 is required for animal development, but whether these proteins also have independent roles in development has been unclear. Known phenotypes associated with loss of either one of these two proteins are very similar and consistent with their joint function, even though both cofactors are involved with additional distinct RNA biogenesis pathways. Here, we report clear phenotypic differences between drosha and pasha/dgcr8 null alleles in two postembryonic lineages in the Drosophila brain: elimination of pasha/dgcr8 leads to defects that are not shared by drosha null mutations in the morphology of gamma neurons in the mushroom body lineage, as well as many neurons in the anterodorsal projection neuron lineage. These morphological defects are not detected in neurons that are genetically depleted of two additional microRNA pathway components, dicer-1 and argonaute1, indicating that they are not due to loss of microRNA activity. They are, however, phenocopied by a newly identified recessive gain-of-function allele in drosha that probably interferes with the microRNA independent functions of Pasha/DGCR8. These data therefore identify a general Drosha-independent DGCR8/Pasha pathway that promotes proper morphology in multiple neuronal lineages. Given that reduction of human DGCR8/Pasha may contribute to the cognitive and behavioral characteristics of DiGeorge syndrome patients, disruption of this newly described pathway could underlie human neurological disease.

ADAR mediates differential expression of polycistronic microRNAs

Adenosine deaminases acting on RNAs (ADARs) convert adenosine residues to inosines in primary microRNA (pri-miRNA) transcripts to alter the structural conformation of these precursors and the subsequent functions of the encoded microRNAs (miRNAs). Here we show that RNA editing by Drosophila ADAR modulates the expression of three co-transcribed miRNAs encoded by the evolutionarily conserved let-7-Complex (let-7-C) locus. For example, a single A-to-I change at the −6 residue of pri-miR-100, the first miRNA in this let-7-C polycistronic transcript, leads to enhanced miRNA processing by Drosha and consequently enhanced functional miR-100 both in vitro as well as in vivo. In contrast, other editing events, including one at the +43 residue of the pri-miR-125, destabilize the primary transcript and reduce the levels of all three encoded miRNAs. Consequently, loss of adar in vivo leads to reduced miR-100 but increased miR-125. In wild-type animals, the destabilizing editing events in pri-let-7-C increase during the larval-to-adult transition and are critical for the normal downregulation of all three miRNAs seen late in metamorphosis. These findings unravel a new regulatory role for ADAR and raise the possibility that ADAR mediates the differential expression characteristic of many polycistronic miRNA clusters.

A let-7-to-miR-125 MicroRNA Switch Regulates Neuronal Integrity and Lifespan in Drosophila

Messenger RNAs (mRNAs) often contain binding sites for multiple, different microRNAs (miRNAs). However, the biological significance of this feature is unclear, since such co-targeting miRNAs could function coordinately, independently, or redundantly with one another. Here, we show that two co-transcribed Drosophila miRNAs, let-7 and miR-125, non-redundantly regulate a common target, the transcription factor Chronologically Inappropriate Morphogenesis (Chinmo). We first characterize novel adult phenotypes associated with loss of both let-7 and miR-125, which are derived from a common, polycistronic transcript that also encodes a third miRNA, miR-100. Consistent with the coordinate upregulation of all three miRNAs in aging flies, these phenotypes include brain degeneration and shortened lifespan. However, transgenic rescue analysis reveal separable roles for these miRNAs: adult miR-125 but not let-7 mutant phenotypes are associated with ectopic Chinmo expression in adult brains and are suppressed by chinmo reduction. In contrast, let-7 is predominantly responsible for regulating chinmo during nervous system formation. These results indicate that let-7 and miR-125 function during two distinct stages, development and adulthood, rather than acting at the same time. These different activities are facilitated by an increased rate of processing of let-7 during development and a lower rate of decay of the accumulated miR-125 in the adult nervous system. Thus, this work not only establishes a key role for the highly conserved miR-125 in aging. It also demonstrates that two co-transcribed miRNAs function independently during distinct stages to regulate a common target, raising the possibility that such biphasic control may be a general feature of clustered miRNAs.

Lin-28 promotes symmetric stem cell division and drives adaptive growth in the adult Drosophila intestine

Stem cells switch between asymmetric and symmetric division to expand in number as tissues grow during development and in response to environmental changes. The stem cell intrinsic proteins controlling this switch are largely unknown, but one candidate is the Lin-28 pluripotency factor. A conserved RNA-binding protein that is downregulated in most animals as they develop from embryos to adults, Lin-28 persists in populations of adult stem cells. Its function in these cells has not been previously characterized. Here, we report that Lin-28 is highly enriched in adult intestinal stem cells in the Drosophila intestine. lin-28 null mutants are homozygous viable but display defects in this population of cells, which fail to undergo a characteristic food-triggered expansion in number and have reduced rates of symmetric division as well as reduced insulin signaling. Immunoprecipitation of Lin-28-bound mRNAs identified Insulin-like Receptor (InR), forced expression of which completely rescues lin-28-associated defects in intestinal stem cell number and division pattern. Furthermore, this stem cell activity of lin-28 is independent of one well-known lin-28 target, the microRNA let-7, which has limited expression in the intestinal epithelium. These results identify Lin-28 as a stem cell intrinsic factor that boosts insulin signaling in intestinal progenitor cells and promotes their symmetric division in response to nutrients, defining a mechanism through which Lin-28 controls the adult stem cell division patterns that underlie tissue homeostasis and regeneration. Highlighted article: Lin-28 boosts insulin signaling in adult Drosophila intestinal progenitors in response to nutrients, adjusting the rate of symmetric renewal of stem cells and driving intestinal tissue growth.

MicroRNAs as components of systemic signaling pathways in Drosophila melanogaster.

MicroRNAs (miRNAs) ensure progression through development by synchronizing cell fate transitions in response to environmental cues. These cues are mediated at least in part by steroid hormones. Emerging evidence indicates that miRNAs are also components of additional systemic signaling pathways, including insulin, stress, immune, and circadian pathways. Thus, the roles that miRNAs play during development are reflected in their post-developmental functions, where they similarly function to coordinate cell behavior in response to environmental cues. In this review, we summarize current work highlighting the role of miRNAs in systemic signaling pathways in Drosophila melanogaster as a way of synthesizing the underlying roles of miRNAs in both animal developmental transitions and adult physiology.