Gene name - mir-14 stem loop
Cytological map position - 54F1
Symbol - mir-14
FlyBase ID: FBgn0262447
Genetic map position -
Classification - microRNA
Cellular location - unknown
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).
Structure of mir-14 is found at Entrez Nucleotide
date revised: 3 September 2003
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