Mir-14 is expressed throughout Drosophila development and in the adult (Sempere, 2003).
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).
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
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
date revised: 3 September 2003
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