InteractiveFly: GeneBrief

mir-14 stem loop: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

MicroRNAs (miRNAs) are small regulatory RNAs that are between 21 and 25 nucleotides in length and repress gene function through interactions with target mRNAs. The genomes of metazoans encode on the order of several hundred miRNAs, but the processes they regulate have been defined for only a few cases. New inhibitors of apoptotic cell death were sought by testing existing collections of P element insertion lines for their ability to enhance a small-eye phenotype associated with eye-specific expression of the Drosophila cell death activator Reaper. The Drosophila miRNA mir-14 has been identified as a cell death suppressor. Loss of mir-14 enhances Reaper-dependent cell death, whereas ectopic expression suppresses cell death induced by multiple stimuli. Animals lacking mir-14 are viable. However, they are stress sensitive and have a reduced lifespan. mir-14 mutants have elevated levels of the apoptotic effector caspase Ice, suggesting one potential site of action. Mir-14 also regulates fat metabolism. Deletion of mir-14 results in animals with increased levels of triacylglycerol and diacylglycerol, whereas increases in mir-14 copy number have the converse effect (Xu, 2003).

Flies that had small eyes due to the eye-specific expression of Reaper (Rpr) (GMR-Rpr flies) were crossed to publicly available lines with a lethal P element insertion on the second chromosome (Bloomington Stock Center) and transheterozygous progeny were examined for enhancement or suppression of the GMR-Rpr small-eye phenotype. One line, l(2)k10213 at 45F1, was identified that was of particular interest. Most l(2)k10213/GMR-Rpr transheterozygotes (87%, n = 300) die as pupae. However, all of those that survived to adulthood showed an enhanced GMR-Rpr small-eye phenotype. These phenotypes persist after the removal of a background lethal mutation on the l(2)k10213 chromosome. However, they are reverted after precise excision of the l(2)k10213 P element, indicating that they are due to the presence of this element. These observations suggested that the 45F1 region contains one or more genes that act as suppressors of Rpr-dependent cell death in multiple contexts-in the eye and in other undefined tissues in which leaky expression of Rpr from the GMR promoter results in organismal death (Xu, 2003).

The annotated protein-coding genes nearest to the l(2)k10213 insertion are CG1888 and CG12931, 6.8 and 5.8 kb away, respectively. However, the noncoding miRNA mir-14 (Lagos-Quintana, 2001) is located only 172 bp from the site of the l(2)k10213 insertion. To explore the possibility that mir-14 functions as a suppressor of Rpr-dependent cell death, imprecise P element excision was used to generate flies carrying a 533 bp deletion encompassing the mir-14 precursor (mir-14Δ¹). GMR-Rpr flies carrying one copy of this deletion show an enhanced small-eye phenotype as well as a high-frequency lethality. Both phenotypes are suppressed when mir-14Δ¹/+;GMR-Rpr/+ heterozygotes carry a 3.4 kb fragment of genomic DNA encompassing the mir-14 region (mir-14^{+3.4 Kb}), consistent with the hypothesis that these phenotypes are due to a loss of mir-14. The mir-14 copy number is also increased by introducing multiple copies of the mir-14^{+3.4 Kb} fragment into a GMR-Rpr background. Increasing the mir-14 dose to three or four copies leads to further suppression of the Rpr-dependent small-eye phenotype (Xu, 2003).

To directly test the hypothesis that mir-14 alone is sufficient to act as a cell death suppressor, flies were generated that expressed a 118 bp fragment of genomic DNA containing the mir-14 precursor under GMR control. The eyes of these flies (GMR-mir-14 flies) were wild-type in appearance. GMR-mir-14 potently suppresses cell death induced by GMR-driven expression of Rpr, Hid, or Grim. Expression of mir-14 also suppresses late-onset retinal-cell death induced by expression of Dronc, an apical caspase that participates in much cell death signaling in the fly. Importantly, however, GMR-mir-14 expression has little or no effect on the eye phenotypes induced by GMR-dependent expression of several other molecules, the long prodomain caspase Strica, whose mechanism of action and normal functions are unknown, or the Ras pathway negative regulator Tramtrack. Together, the results of these loss- and gain-of-function experiments argue that mir-14 is a dose-dependent suppressor of Rpr-, Hid-, Grim-, and Dronc-dependent cell death (Xu, 2003).

These studies have shown that mir-14 suppresses death induced by expression of either Rpr, Hid, Grim, or the apical caspase Dronc. Furthermore, loss of mir-14 enhances Rpr-dependent cell death, suggesting that mir-14 normally participates in death inhibition in some contexts. From gene activation screens, several other miRNAs have been identified that suppress cell death when ectopically expressed in the fly eye (unpublished data cited in Xu, 2003). Together, these observations suggest that miRNAs are likely to constitute a heretofore hidden resource of cell death regulators. The identification of miRNAs that inhibit cell death is important for several reasons. It broadens the contexts in which miRNAs are known to function. In addition, it defines new points and mechanisms of cell death regulation. The identification of cell death-regulating miRNAs may also be important for understanding how cell survival is regulated in human disease. For example, it is likely that death-inhibiting miRNAs, being very small and noncoding, would not have been identified in previous screens for genes that promote oncogenesis by inhibiting cell death. They would also have been missed in experiments designed to identify candidate oncogenes through transcriptional profiling of normal and transformed cells because these experiments were not designed to detect miRNAs. Thus, it is reasonable to propose that deregulation of miRNA expression may contribute to the inappropriate survival that is so important for oncogenic progression (Xu, 2003).

Mir-14 dosage also regulates the levels of organismal DAG and TAG. DAG and TAG synthesis, storage, utilization, and degradation are regulated at many levels depending on cell type, as well as energy and signaling needs. Targets for mir-14 as a regulator of fat metabolism may be distinct from those that mediate its role as a cell death inhibitor. However, a number of described links between fat metabolism and apoptotic signaling suggest ways in which these phenotypes might be related. Overnutrition-induced obesity, lipodystrophy, and type II diabetes, as well as defects in TAG β-oxidation, lead to the accumulation of long-chain fatty acids in the form of TAG. TAG itself is probably not toxic. However, particularly in cells other than adipocytes, the surplus fatty acyl CoA can enter other nonoxidative pathways that promote cell dysfunction and/or cell death, including a form of caspase-dependent cell death known as lipoapoptosis. Lipoapoptosis is driven, at least in part, by the de novo production of ceramide from excess fatty acyl CoA. Rpr expression also promotes de novo production of ceramide, and there is evidence that ceramide plays a role in mediating some of Rpr's pro-apoptotic effects. It will be interesting to determine if mir-14 regulates the levels of fatty acyl CoA precursors or enzymes required for de novo ceramide synthesis. Also, DAG is an important second messenger in multiple signal transduction pathways, some of which are linked to apoptosis induction. DAG-dependent signals are terminated by mechanisms that remove DAG. Mobilization of DAG into cellular TAG stores by DGAT is one quantitatively important pathway by which this is brought about. Drosophila encodes multiple genes with homology to mammalian DGATs (CG31991, CG1941, and CG1942). Interestingly, mutations in the Drosophila gene midway (mdy, CG31991), which encodes a DGAT expressed predominantly in the female germline, led to decreased nurse cell TAG levels and premature nurse cell death. The mechanism by which mdy mutations promote nurse cell death is unknown. However, it is intriguing to speculate that loss of mdy, and perhaps mir-14 as well, leads to an increase in the levels of unesterified intracellular DAG and thereby promotes cell death signaling. An important question for the future will be whether regulation of mir-14 transcription or processing serves as a point of control for cell death, stress sensitivity, or fat storage in different environmental conditions or behavioral states (Xu, 2003).

REGULATION

microRNA miR-14 acts to modulate a positive autoregulatory loop controlling steroid hormone signaling in Drosophila

The insect steroid hormone Ecdysone and its receptor play important roles during development and metamorphosis and regulate adult physiology and life span. Ecdysone signaling, via the Ecdysone receptor (EcR), has been proposed to act in a positive autoregulatory loop to increase EcR levels and sensitize the animal to ecdysone pulses. Evidence is presented that this involves EcR-dependent transcription of the EcR gene, and that the microRNA miR-14 modulates this loop by limiting expression of its target EcR. Ecdysone signaling, via EcR, down-regulates miR-14. This alleviates miR-14-mediated repression of EcR and amplifies the response. Failure to limit EcR levels is responsible for the many of the defects observed in miR-14 mutants. miR-14 plays a key role in modulating the positive autoregulatory loop by which Ecdysone sensitizes its own signaling pathway (Varghese, 2007).

These experiments provide evidence that Ecdysone signaling reciprocally regulates transcription of the miR-14 and EcR genes. Thus ecdysone acts in two ways to induce EcR activity: first by promoting EcR transcriptional autoregulation and second by alleviating miR-14-mediated repression of EcR activity. Prior to an ecdysone pulse the balance between EcR autoinduction and the mutually repressive interaction between EcR and miR-14 will keep a stable low level of EcR activity. On ecdysone stimulation, the balance shifts to a higher level of EcR activity, but to do so it must overcome repression by miR-14. In the absence of miR-14 one means to limit EcR autoinduction loop is lost, and defects result due to excess EcR activity (Varghese, 2007).

This 'belt and suspenders' approach to EcR regulation may be needed because of the intrinsic lability of a positive autoregulatory mechanism based on a simple transcriptional loop. Such systems can permit a sharp switch-like response, but stochastic variation in EcR levels due to transcriptional 'noise' could trigger the autoregulatory loop at random. The requirement to overcome miR-14-mediated repression thus provides a buffer. Transient fluctuations in EcR levels will transiently repress miR-14 transcription, but the existing miR-14 would take some time to decay. Therefore a transient 'noise' in EcR levels is unlikely to overcome miR-14-mediated repression. In contrast, a sustained induction of EcR by ecdysone allows for decay of miR-14 and sustained autoregulation (Varghese, 2007).

Most miRNAs are predicted to have hundreds of potential target genes, and often the predicted target sites are conserved in evolution, providing some confidence that they are functional. Yet in several cases much of the biological output of the miRNA, as assessed by its mutant phenotype, has been linked to only one or a few of the predicted targets. miR-14 is no exception, with ~180 predicted targets. It is noted that EcR misregulation accounts for only some of the defects in the miR-14 mutants, indicating that other potential targets may play important roles mediating the effects of miR-14 in other contexts (Varghese, 2007).

The relationship between miR-14 and EcR is similar in some respects to that of miR-9a, and the positive feedback loop involved in sense organ specification and senseless expression in Drosophila. The proneural basic helix-loop-helix (bHLH) transcription factors form a positive regulatory loop with the zinc-finger transcription factor Senseless. miR-9a sets a threshold of activity that must be exceeded for the loop to activate by limiting Senseless expression. When coupled with a positive autoregulatory circuit, miRNAs can provide an effective means by which to set thresholds and limit noise-induced errors to ensure robustness in development (Varghese, 2007).

Drosophila miR-14 regulates insulin production and metabolism through its target, sugarbabe

Energy homeostasis depends on insulin signaling in metazoans. Insulin levels reflect the nutritional status of the animal to control levels of circulating sugar and regulate storage of resources in the form of glycogen and fat. Over the past several years, evidence has begun to accumulate that insulin production and secretion, as well as cellular responsiveness to insulin, are subject to regulation by microRNAs. This study presents evidence that miR-14 acts in the insulin-producing neurosecretory cells in the adult Drosophila brain to control metabolism. miR-14 acts in these cells through its direct target, sugarbabe. sugarbabe encodes a predicted zinc finger protein that regulates insulin gene expression in the neurosecretory cells. Regulation of sugarbabe levels by nutrients and by miR-14 combines to allow the fly to manage resource mobilization in a nutritionally variable environment (Varghese, 2010).

In mice, miR-375 affects metabolism by regulating insulin secretion in pancreatic β cells. Mutant mice lacking miR-375 show defects in glucose homeostasis, apparently due to reduced pancreatic β-cell mass. Mammalian microRNAs (miRNAs) also regulate insulin levels by other means. miR-9 and miR-96 have been implicated in insulin release through up-regulation of granuphilin, a negative regulator of secretion involved in vesicle docking (Varghese, 2010).

In Drosophila, miRNAs have been implicated in regulating insulin responsiveness in a metabolically important organ called the fat body. The fat body combines the functions of liver and adipose tissue and plays a central role in coordinating metabolism and growth of the organism during development. miR-278 mutant flies show insulin resistance in most tissues, but most strongly in the fat body (Teleman, 2006). miR-278 acts by regulating expression of the expanded gene. Expanded is best known as a membrane-associated FERM domain protein, which negatively regulates the Hippo signaling pathway. However, the Hippo pathway does not appear to be involved in this context. A second miRNA, miR-8, also acts in fat body to control insulin signaling (Hyun, 2009). miR-8 and its vertebrate ortholog, miR-200, indirectly activate phosphatidylinositol 3-kinase (PI3K) by repressing the expression of their conserved targets, U-shaped/FOG2. FOG2 protein binds to the regulatory subunit of PI3K and prevents formation of the active enzyme complex. Thus, loss of mir-8 leads to reduced insulin sensitivity in the fat body, with effects on growth and metabolism (Varghese, 2010).

In Drosophila, metabolic control by insulin production depends on insulin gene expression in a set of 14 neurosecretory cells in the brain. The developmental origin of these insulin-producing cells (IPCs) suggests an evolutionary relationship to mammalian pancreatic β cells. Flies lacking IPCs have elevated carbohydrate and fat levels, indicating a role for IPCs in metabolic control. Previous reports have shown that miR-14 mutant flies present defects related to apoptosis, stress response, survival, and metabolism (Xu, 2003). Misregulation of the Ecdysone receptor (EcR) was shown to be the cause of the pupal stage survival and metamorphosis defects and the reduced adult life span, but was not responsible for the obesity observed in the mutant adult flies (Varghese, 2007). This study provides evidence that the metabolic function of miR-14 depends on its activity in the IPCs, and explores its mechanism of action (Varghese, 2010).

To explore the basis for the metabolic defect in miR-14 mutant flies, tissue-specific rescue of the mutant phenotype was used as a means to determine where miR-14 expression is required. It was first confirmed that expression of a UAS-miR-14 transgene under the control of a ubiquitously expressed Gal4-driver (armadillo-Gal4) could rescue the obesity phenotype. To assess obesity, the ratio of total body triglyceride was compared with total body protein. miR-14 mutant flies showed an elevated fat to protein ratio, but this was restored to normal in the rescued mutant. Interestingly, the mutant was not rescued by expressing UAS-miR-14 in the adipose tissue, using the fat body-specific driver lsp2-Gal4. This finding suggested that miR-14 does not act in the adipose tissue to regulate triglyceride levels (Varghese, 2010).

Use was made of the observation that miR-14 overexpression can make flies lean (Xu, 2003) to identify tissues in which miR-14 activity can influence fat levels. Expression of miR-14 in the CNS using a pan-neuronal driver -- or, more selectively, in the neurosecretory IPCs using dILP2-Gal4 -- produced lean flies. A miR-14 lac-Z reporter transgene showed expression in the IPCs and in most other cells of the brain (IPCs are identified by dilp-Gal4 UAS-nRFP). Expression of a miR-14 sensor (a ubiquitously expressed GFP reporter transgene with perfect miR-14 sites in the 3' untranslated region [UTR]) allows for strong down-regulation of the sensor in miR-14-expressing cells. This is illustrated by comparing sensor levels in miR-14 mutant clones and neighboring miR-14/+ heterozygous tissue in larval wing discs. Similarly, miR-14 sensor levels were very low in control brains, but increased considerably in miR-14 mutant brains, indicating that miR-14 is active in the IPCs as well as other cells of the brain. Restoring miR-14 expression selectively in the IPCs of otherwise miR-14 mutant flies proved to be sufficient to restore fat levels to normal. Although miR-14 is broadly expressed and active in the brain, this tissue-specific rescue identifies the IPCs as a key site of miR-14 function in the control of metabolism (Varghese, 2010).

How does miR-14 expression in the IPCs affect fat levels? Previous work has shown that systemic insensitivity to insulin leads to an obese phenotype. Flies lacking IPCs or with reduced insulin-like peptide (ILP) levels store excess fat. Therefore expression of the genes encoding IPC-specific ILPs was examined in the miR-14 mutant. The nutrient-sensitive ilp (ilp3 and ilp5) mRNA levels were reduced. This was partially rescued by miR-14 expression in the mutant IPCs. The level of the non-nutrient-sensitive ilp2 mRNA was also reduced, but to a lesser extent. Interestingly, restoring ilp3 expression in the IPCs by expression of a UAS-ilp3 transgene was sufficient to restore normal fat levels in the miR-14 mutant. This suggests that reduced ilp gene expression in the IPCs is sufficient to explain the metabolic effects of removing miR-14 (Varghese, 2010).

The observation that IPC-specific expression was sufficient to rescue the miR-14 phenotype provided a means to search for miR-14 targets that are functionally relevant in these cells. Expression profiling was performed using RNA isolated from adult heads of control flies, miR-14 mutants, and IPC-rescued miR-14 mutants. Of 810 mRNAs up-regulated by >1.5-fold in the mutant, 165 were reduced by >50% in the mutant with miR-14 expression restored selectively in the IPCs. Forty-six of these had predicted miR-14 target sites in their 3'UTRs. Ten of the 46 were among mRNAs enriched by Ago1 immunoprecipitation from S2 cells, where miR-14 is abundantly expressed. These 10 mRNAs were selected for further analysis (Varghese, 2010).

dILP2-Gal4 was used to express UAS-RNAi transgenes to reduce expression of the 10 candidates in the IPCs. This should mimic the effects of miR-14 overexpression, which makes flies lean. Two of the candidates, sugarbabe and fuzzy, produced a lean phenotype when depleted by RNAi. As a second test, it was asked whether overexpression in the IPCs could mimic the miRNA mutant phenotype. Overexpression of fuzzy had no effect, but sugarbabe overexpression in the IPCs led to elevated fat levels and reduced levels of ilp transcripts. Similar to what was observed in the miR-14 mutant flies, the effect of sugarbabe overexpression was more pronounced on the levels of the two nutrient-sensitive ilp transcripts (Varghese, 2010).

These experiments suggest that sugarbabe might be a biologically relevant target of miR-14 activity in the IPCs. Fluorescent in situ hybridization (FISH) was used to visualize sugarbabe mRNA expression in the adult brain. sugarbabe mRNA Is present in the IPC cells (identified by dILP2-Gal4-directed expression of a UAS-nRFP transgene). Little or no sugarbabe mRNA was detected in other parts of the brain (Varghese, 2010).

As a more stringent test of sugarbabe function, it was asked whether limiting its overexpression selectively in the IPCs would reduce the severity of the miR-14 mutant phenotype. IPC-specific expression of a sugarbabe UAS-RNAi transgene reduced sugarbabe mRNA levels in the adult head by ~30% and resulted in increased ilp transcript levels. sugarbabe transcript levels increased approximately threefold in miR-14 mutant head RNA, and this was partially offset by IPC-specific expression of the UAS-RNAi transgene. This also lowered fat levels. These assays point to sugarbabe as a functionally important miR-14 target in the IPCs (Varghese, 2010).

There is one predicted miR-14 site in the sugarbabe 3'UTR. This site is conserved in the eight species of the Drosophilia melanogaster and obscura groups. To test its function in vivo, transgenic flies were prpared expressing a GFP reporter with the intact sugarbabe 3'UTR. GFP expression levels were higher in the IPCs and in other brain cells in miR-14 mutant flies compared with wild-type controls. This indicates that endogenous levels of miR-14 are sufficient to negatively regulate gene expression via the sugarbabe 3'UTR in the IPCs in vivo. As a further test, use was made of a luciferase reporter with the intact sugarbabe 3'UTR and a second version in which the site was altered to disrupt pairing to the miRNA seed. Because S2 cells endogenously express miR-14, it was asked whether depleting miR-14 would lead to elevated expression of the luciferase reporters. The reporter with the mutated site was unchanged, but the reporter with the intact miR-14 site showed a 40% increase in activity in miR-14-depleted cells. Reciprocally, overexpression of miR-14 caused reduced luciferase activity from the intact reporter. These results suggest that miR-14 can act directly via the predicted target site to regulate sugarbabe levels (Varghese, 2010).

In the laboratory, flies are reared on a nutritionally rich medium containing protein, amino acids, fats, and complex sugars. Natural environments offer a more variable nutrient supply, and the ability to adapt to changing conditions may confer an advantage. Like other animals, flies store energy in the form of glycogen and fat. The contribution of miR-14 and sugarbabe to their response to nutrient stress was examined. For these experiments, larvae were reared under controlled growth conditions on rich medium, and adult flies were aged on this medium for 4 d before being deprived of nutrients. miR-14 mutant flies were more sensitive to nutrient deprivation, as were flies selectively overexpressing sugarbabe in the IPCs, which phenocopies the miR-14 mutant. It was confirmed that the reduced survival of the miR-14 mutants under these conditions was due to elevated sugarbabe levels by selectively expressing the sugarbabe RNAi transgene in the IPC cells of the miR-14 mutant. Partially compensating for the elevated sugarbabe mRNA level improved survival of the mutant flies (Varghese, 2010).

At the start of the experiment, before nutrient deprivation, fat levels were higher in the mutant, but glycogen levels were lower - an expected outcome of reduced insulin signaling. During nutrient deprivation, glycogen drops more quickly than in control flies, but reaches similar low levels by 36 h. Fat reserves were initially mobilized more slowly, but then dropped sharply at the stage when the mutant flies begin to die. Thus, there is an initial metabolic imbalance in the miR-14 mutants, with nutrient reserves shifted more toward fat storage, perhaps at the expense of glycogen. The resulting imbalance in nutrient mobilization may be responsible for the impaired survival of the mutant flies when nutrient-deprived (Varghese, 2010).

Overexpression of sugarbabe in the IPCs is sufficient to mimic the effects of the miR-14 mutant on fat storage, expression of the nutritionally sensitive ilp transcripts, and starvation sensitivity. To further explore their function in this context, it was asked whether their expression in the adult head was nutrient-responsive. sugarbabe has been shown previously to be up-regulated in the gut, malphigian tubules, and fat body tissues of larvae fed on a high-sugar diet (Zinke, 2002). It was found that sugarbabe mRNA levels decreased by >50% in RNA from adult heads, whereas miR-14 levels were not affected significantly. The change in sugarbabe levels was also seen in nutrient-deprived miR-14 mutants, showing a miR-14-independent change of sugarbabe during starvation. Thus, sugarbabe expression is controlled in the IPCs by two independent means (Varghese, 2010).

Sugarbabe encodes a predicted zinc finger protein (Zinke, 2002). The current findings suggest that sugarbabe levels in the IPC are under nutritional regulation, and that this in turn controls the levels of ilp mRNAs. Sugarbabe might act as a transcription factor to regulate ilp gene expression, but the possibility cannot be excluded that its effects are post-transcriptional, affecting RNA processing or stability (Varghese, 2010).

How do the nutrition-dependent changes in sugarbabe expression correlate with regulation of ilp transcript levels? Based on the data presented so far, the observed reduction in sugarbabe levels upon nutrient deprivation is expected to elevate ilp transcript levels. Yet, ilp levels decrease under these conditions. To address this issue, the effects were examined of offsetting the decrease in sugarbabe levels by expressing UAS- sugarbabe in the IPCs under dilp2-Gal4 control. Under these conditions, the levels of ilp3 and ilp5 transcripts decreased by considerably more than in the nutrient-deprived controls. This indicates that down-regulation of sugarbabe during nutrient deprivation serves to limit the effects of nutrient deprivation on ilp gene expression (Varghese, 2010).

The findings highlight the importance of sugarbabe levels in the IPCs as a regulator of energy balance. Under normal circumstances, elevated fat levels can confer resistance to starvation. However, flies with defects in fat mobilization can be hypersensitive to starvation despite being obese. Impairing insulin signaling has been shown to enhance fat storage, but it can also sensitize flies to starvation in some contexts. miR-14 mutants show a similar syndrome of low insulin and obesity coupled with starvation sensitivity. The findings suggest that nutritional regulation of sugarbabe might serve to limit the reduction of insulin expression during starvation. It is speculated that maintaining a sufficient level of insulin activity might be important to allow mobilization of nutrient reserves from fat stores. The finding that sugarbabe is nutritionally controlled, while miR-14 levels are not, provides the opportunity for an interplay between nutritionally dependent and independent regulation of insulin production (Varghese, 2010).

DEVELOPMENTAL BIOLOGY

Mir-14 is expressed throughout Drosophila development and in the adult (Sempere, 2003).

EFFECTS OF MUTATION

Loss of a cell death suppressor might be expected to result in reduced organismal viability and/or increased stress sensitivity. Homozygous mir-14Δ¹ embryos from heterozygous parents hatch at a normal frequency, and larvae survive to pupal stages at a rate similar to that of the wild-type. However, most mir-14 larvae die during pupal development. Eclosion of those that survive is somewhat delayed with respect to the wild-type. Both of these phenotypes are reverted when mir-14Δ¹ homozygotes carry two copies of the mir-14^{+3.4 Kb} genomic fragment. mir-14Δ¹ homozygous adults also have a decreased mean and maximal lifespan. This decrease is particularly marked in females. Finally, homozygous mir-14Δ¹ larvae were also much more susceptible than wild-type larvae to being killed by salt stress, an activator of the p38 mitogen-activated protein kinase (MAPK) pathway in Drosophila. This phenotype is rescued by the presence of the mir-14^{+3.4 Kb} fragment. In summary, flies lacking mir-14 show compromised viability in multiple assays (Xu, 2003).

The two C. elegans miRNAs with known functions, lin-4 and let-7, are thought to regulate development by binding to the 3'untranslated region of target transcripts and thereby repressing the translation of their products. In these examples, the analysis of genetic interactions provides important clues as to the identity of targets. In the absence of this sort of information, it is difficult to predict miRNA targets in animals. This is because base pairing between the mature miRNA and its target is imperfect and the rules that govern which base pair interactions are important are unknown. Potential Mir-14 binding sites were sought in a number of apoptotic regulators, including Dronc, Rpr, Hid, and Grim. Potential target sites were identified in the transcripts of several genes, including Ice, Dcp-1, Scythe, SkpA, and Grim (however, the Grim target is present in the 3'UTR, which was absent in the GMR-Grim transgene). Of these, Ice, an apoptotic effector caspase, is of particular interest. Ice is required for at least some cell deaths and is activated by Dronc, which promotes cell death induced by Rpr, Hid, and Grim. Ice levels in adults were measured by using an anti-Ice antibody. Ice is elevated in mir-14Δ¹ flies as compared to the wild-type, and this increase is suppressed in the presence of two copies of the mir-14-containing 3.4 kb genomic DNA fragment. Whereas these observations alone do not prove that Ice is a direct target of Mir-14, they do suggest that Ice is regulated, either directly or indirectly, by Mir-14 levels (Xu, 2003).

Plastic sections were examined from adult wild-type and mir-14Δ¹ flies. The overall cellular architecture of heads from mir-14Δ¹ flies was normal. However, one striking phenotype was noted. Adipocyte lipid droplets are greatly enlarged in mir-14Δ¹ flies. This phenotype is suppressed in the presence of two copies of the mir-14-containing 3.4 kb genomic DNA fragment, consistent with the hypothesis that it is due to loss of mir-14 (Xu, 2003).

Triacylglycerols (TAG) are the major component of adipocyte lipid droplets. All cells store small amounts of TAG that participate in phospholipid synthesis. However, adipocytes are the primary site of storage for an organism's overall needs. This suggests that mir-14Δ¹ adults might have elevated levels of TAG. In fact, the TAG content of mir-14Δ¹ adults is increased about 2-fold over that of wild-type flies. Dietary fats in Drosophila are transported from the midgut to their major storage site in the fat body as diacylglycerol (DAG) bound to a lipoprotein particle. At the fat body, DAG is converted into lipid droplet TAG for storage by the activity of an acyl CoA:diacylglycerol transferase (DGAT). Lipids are also mobilized from the fat body as DAG. TAG lipases produce DAG from TAG inside the adipocyte. DAG is then transported to the cell surface and added to a lipoprotein particle that is transported to target tissues through the hemolymph. Diacylglycerol is also a precursor for the synthesis of multiple phospholipids and an important second messenger in multiple signal transduction pathways. Interestingly, diacylglycerol levels are also significantly elevated in mir-14Δ¹ flies. Flies that carry four copies of mir-14, two endogenous copies and two copies of the mir-14^{+3.4 Kb} fragment, have a set of phenotypes that are the converse of the phenotype of animals lacking mir-14-a decrease in diacylglycerol and triacylglycerol levels. Levels of several other lipid classes, including free fatty acids, cholesterol esters, lysophosphatidylcholine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, and total phospholipids are not significantly changed in flies lacking mir-14 or carrying additional copies of mir-14. Together, these observations argue that mir-14 is a dose-dependent regulator of DAG and TAG metabolism in Drosophila (Xu, 2003).

REFERENCES

Search PubMed for articles about Drosophila mir-14 stem loop

Hyun, S., Lee, J. H., Jin, H., Nam, J., Namkoong, B., Lee, G., Chung, J. and Kim, V. N. (2009). Conserved MicroRNA miR-8/miR-200 and its target USH/FOG2 control growth by regulating PI3K. Cell 139: 1096-1108. PubMed Citation: 20005803

Lagos-Quintana, M., Rauhut, R., Lendeckel, W., and Tuschl, T. (2001). Identification of novel genes coding for small expressed RNAs. Science 294: 853-858. 11679670

Sempere, L.F., Sokol, N.S., Dubrovsky, E.B., Berger, E.M., and Ambros, V. (2003). Temporal regulation of microRNAs expression mediated by hormonal inputs and broad-complex pupal-specific transcription factor in Drosophila melanogaster. Dev. Biol. 259(1): 9-18. 12812784

Teleman, A. A., Maitra, S. and Cohen, S. M.. (2006). Drosophila lacking microRNA miR-278 are defective in energy homeostasis. Genes Dev 20: 417-422. PubMed Citation: 16481470

Varghese, J. and Cohen, S. M. (2007). microRNA miR-14 acts to modulate a positive autoregulatory loop controlling steroid hormone signaling in Drosophila. Genes Dev. 21(18): 2277-82. PubMed Citation: 17761811

Varghese, J., Lim, S. F. and Cohen, S. M. (2010). Drosophila miR-14 regulates insulin production and metabolism through its target, sugarbabe. Genes Dev. 24(24): 2748-53. PubMed Citation: 21159815

Xu, P., Vernooy, S. Y., Guo, M. and Hay, B. A. (2003). The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism. Curr. Biol. 13: 790-795. 12725740

Zinke, I., et al. (2002). Nutrient control of gene expression in Drosophila: Microarray analysis of starvation and sugar-dependent response. EMBO J 21: 6162-6173. PubMed Citation: 12426388