Inhibition of micro-RNA-induced RNA silencing by 2'-o-methyl oligonucleotides in Drosophila S2 cells.

More than 90 different micro-ribonucleic acid (miRNA) encoding genes have been identified in Drosophila, yet the function of only two of these, bantam and DmiR-14, has been elucidated. In an effort to develop a general strategy for the analysis of miRNA function in Drosophila, two procedures were developed, in a Schneider line 2 cell culture system, which may be adapted to that end. First, we show that endogenous miRNAs can partially inhibit the expression of a transiently transfected reporter gene that has been modified to contain sequences complementary to that miRNA in the 3' UTR of a target messenger RNA (mRNA). Inhibition occurs by RNA interference (RNAi), which involves mRNA degradation. Second, we demonstrate that this miRNA-induced RNAi can be partially rescued with 2'-O-methyl oligonucleotides that contain sequences complementary to the cognate miRNA. We discuss how these techniques may be used, in vivo, both for localizing the tissue distribution of endogenous miRNAs during Drosophila development and identifying phenotypes associated with a loss of miRNA function.

Juvenile hormone regulation of the E75 nuclear receptor is conserved in Diptera and Lepidoptera.

Despite longstanding efforts, the juvenile hormone (JH) signaling pathway remains unknown. In Drosophila melanogaster (Diptera), JH activates expression of the E75A nuclear receptor. The E75 gene encodes a family of related proteins. A homologue of Drosophila E75 was previously identified and two isoforms, mE75A and mE75B, were reported in Manduca sexta (Lepidoptera). Here, we describe the identification of two additional isoforms, mE75C and mE75D, and the hormonal regulation of mE75 gene expression in Manduca CH1 cultured cells. mE75A and mE75B isoforms are specifically induced by ecdysone in CH1 cells. One isoform, mE75C, shows constitutive expression. The mE75D isoform exhibits dual hormonal regulation; it can be activated by either ecdysone or the JH analog, methoprene. E75-encoded proteins represent the first example of transcription factors directly induced by JH. E75 activation by JH, in both Diptera and Lepidoptera, suggests a conserved function in the JH signaling pathway.

Hormonal regulation and functional role of Drosophila E75A orphan nuclear receptor in the juvenile hormone signaling pathway.

Ecdysone and juvenile hormone (JH) are important regulators of insect growth and development. While ecdysone initiates a transition from one developmental stage to another, JH determines the nature of the transition. How these two hormones interact at the molecular level is not known. Here we report the JH inducibility of the E75A nuclear receptor encoded by the E75 early ecdysone-inducible gene. In Drosophila S2 cells, E75A transcription is specifically activated by JH at concentrations well within the physiological range found in larvae and adults. The induction is rapid and does not require a concurrent protein synthesis, and thus represents a primary hormone response. Consistent with JH regulation, E75A mRNA levels are reduced in ovaries of apterous(4) mutant adults defective in JH secretion. Expression is rescued by topical methoprene application. We further provide evidence that ectopic E75A is sufficient to perform several functions in the JH signaling pathway. First, it can down-regulate its own transcription. Second, E75A can potentiate the JH inducibility of a secondary response gene, JhI-21. Finally, in the presence of JH, E75A can repress ecdysone activation of early genes including Broad-Complex. Based on these data, we propose a model for the role of E75A in the ecdysone-JH regulatory interplay.

Temporal regulation of microRNA expression in Drosophila melanogaster mediated by hormonal signals and broad-Complex gene activity.

lin-4 and let-7 are founding members of an extensive family of genes that produce small transcripts, termed microRNAs (miRNAs). In Caenorhabditis elegans, lin-4 and let-7 control the timing of postembryonic events by translational repression of target genes, permitting progression from early to late developmental programs. To identify Drosophila melanogaster miRNAs that could play similar roles in the control of developmental timing, we characterized the developmental expression profile of 24 miRNAs in Drosophila, and found 7 miRNAs that are either upregulated or downregulated in conjunction with metamorphosis. The upregulation of three of these miRNAs (mir-100, mir-125, and let-7), and the downregulation of a fourth (mir-34) requires the hormone ecdysone (Ecd) and the activity of the Ecd-inducible gene Broad-Complex. Interestingly, mir-125 is a putative homologue of lin-4. mir-100, -125, and let-7 are clustered within an 800-bp region on chromosome 2L, suggesting that these three miRNAs may be coordinately regulated via common cis-acting elements during metamorphosis. In S2 cells, Ecd and the juvenile hormone analog methoprene exert opposite effects on the expression of these four miRNAs, indicating the participation of both these hormones in the temporal regulation of mir-34, -100, -125, and let-7 expression in vivo.

The expression of the let-7 small regulatory RNA is controlled by ecdysone during metamorphosis in Drosophila melanogaster.

In Caenorhabditis elegans, the heterochronic pathway controls the timing of developmental events during the larval stages. A component of this pathway, the let-7 small regulatory RNA, is expressed at the late stages of development and promotes the transition from larval to adult (L/A) stages. The stage-specificity of let-7 expression, which is crucial for the proper timing of the worm L/A transition, is conserved in Drosophila melanogaster and other invertebrates. In Drosophila, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) control the timing of the transition from larval to pupal to adult stages. To test whether let-7 expression is regulated by ecdysone in Drosophila, we used Northern blot analysis to examine the effect of altered ecdysone levels on let-7 expression in mutant animals, organ cultures, and S2 cultured cells. Experiments were conducted to test the role of Broad-Complex (BR-C), an essential component in the ecdysone pathway, in let-7 expression. We show that ecdysone and BR-C are required for let-7 expression, indicating that the ecdysone pathway regulates the temporal expression of let-7 in Drosophila. These results demonstrate an interaction between steroid hormone signaling and the heterochronic pathway in insects.