Computational studies have identified the Ubx 3' UTR as a likely target of regulation by iab-4-5p (Stark, 2003; Grun, 2005). Of the seven potential sites identified by Stark, five exhibit conserved and canonical seed pairing of six or more nucleotides. Of these, sites #3 and #6 are perfectly conserved among sequenced Drosophilids and have seeds of at least 7 nt, a length sufficient for efficient in vivo recognition by miRNAs; site #7 also has a 7-mer seed match that is conserved in some species (Ronshaugen, 2005).
In current target-finding approaches, greater confidence is usually ascribed to those miRNA-binding sites that are conserved in the greatest number of analyzed species. Curiously, the putative iab-4-5p target sites with the lowest free energy are not necessarily the best conserved. Instead, there appear to be compensatory changes among different iab-4-5p-binding sites in individual Ubx 3' UTRs. For example, site #4 exhibits canonical 6-mer seed pairing in four species of Drosophila, but contains a G:U base pair in Drosophila virilis and a seed mismatch in Drosophila mojavenesis and is likely nonfunctional in these two species. Conversely, site #7 is mispaired in D. melanogaster and Drosophila yakuba, but is conserved as a strong 7-mer seed-paired site in D. mojavenesis, Drosophila pseudoobscura, Drosophila ananassae, and D. virilis. These observations suggest that individual target sites may be evolutionarily labile, and in vivo regulation depends on the net complement of both high- and low-affinity sites contained in the target mRNA. These compensatory changes in strong and weak target sites are reminiscent of the evolution of individual Bicoid-binding sites in the eve stripe 2 enhancers present in divergent Drosophilids (Ronshaugen, 2005).
Direct evidence for iab-4:Ubx miRNA interactions was obtained using a tub::GFP-Ubx 3' UTR transgene (the "Ubx sensor"). This construct directs ubiquitous expression of the GFP coding sequence fused to the Ubx 3' UTR, and wing imaginal discs bearing the Ubx sensor display relatively uniform expression of GFP. Ectopic expression of UAS-DsRed under the control of ptc-Gal4, which directs expression along the anterior-posterior border of the disc, has little or no effect on the distribution of GFP staining (Ronshaugen, 2005).
The expression of the Ubx sensor was assayed in the presence of ectopic iab-4 miRNAs. For this purpose, a transgene was created that contains DsRed and 400 base pairs (bp) from iab-4 encompassing the entire 100-bp 3' hairpin sequence (UAS-DsRed-iab-4). Transgenes of this type direct the expression of biologically active miRNAs in cells that are labeled by expression of DsRed (Stark, 2003). When driven by ptc-Gal4 in wing imaginal discs, Ubx sensor levels were specifically diminished in those cells expressing the iab-4 transgene. Detailed analysis of the DsRed-iab-4 and GFP-Ubx expression profiles suggests that repression of the Ubx sensor by ectopic iab-4 miRNA is dose-sensitive. These data constitute in vivo evidence that iab-4 miRNAs specifically recognize target sequences in the Ubx 3' UTR and thereby attenuate Ubx protein synthesis (Ronshaugen, 2005).
Ubx protein is broadly distributed throughout the haltere imaginal disc, where it imposes haltere identity by repressing the expression of many genes that otherwise direct wing development. This repression is very sensitive to Ubx levels, and consequently, even partial loss of Ubx function can transform halteres into wings. Haltere discs were examined for the accumulation of Ubx protein in the absence or presence of ectopic iab-4 miRNAs. Ubx is detected at high levels in most of the cells of the presumptive pouch. Expression of DsRed alone using bx-Gal4, which is active in the presumptive dorsal region of the pouch, did not affect Ubx accumulation. In contrast, haltere discs expressing UAS-DsRed-iab-4 under the control of bx-Gal4 displayed strongly reduced levels of Ubx protein. Thus, as seen for the Ubx sensor in wing discs, ectopic iab-4 miRNA inhibits accumulation of endogenous Ubx protein (Ronshaugen, 2005).
The effect of iab-4 miRNA misexpression on adult haltere development was examined. The wild-type haltere contains small lightly pigmented sensilla but lacks the triple row of sensory bristles at the leading margin seen in wings. In contrast, halteres that developed from discs expressing UAS-DsRed-iab-4 under the control of bx-Gal4 or scalloped-Gal4 are flattened and elongated in the proximal-distal axis, and exhibit an extensive row of sensory bristles at the leading margin. All of these phenotypes are strongly indicative of a classic haltere-to-wing homeotic transformation (Ronshaugen, 2005).
The demonstration that miR-iab-4 represses the anterior Hox gene Ubx might be relevant to the phenomenon of 'posterior prevalence'. Polycomb mutant embryos have previously been observed to derepress Hox gene expression, resulting in broad misexpression of all Hox genes. Ultimately, ectopic expression of posterior Hox genes (e.g., Abd-B or Hox9-13) leads to the transcriptional repression of anterior Hox genes (e.g., Ubx or Hox8 paralogs). Polycomb mutant embryos also derepress iab-4 expression throughout the embryo. Therefore, misexpression of iab-4 miRNAs may contribute to the repression of Ubx function observed in the Polycomb mutant background. Thus, posterior prevalence may arise from the dual utilization of protein-based/transcriptional mechanisms and miRNA-based/post-transcriptional mechanisms (Ronshaugen, 2005).
Double-label RNA FISH and antibody staining was used to determine the relative expression patterns of iab-4 RNA and Ubx RNA/protein accumulation during embryonic development. The iab-4 primary transcript is strongly expressed in the presumptive abdomen, mainly in the progenitors of the second (A2) through seventh (A7) segments (see Cumberledge, 1990). There is also a weak transient stripe of expression in anterior regions. Ubx RNA is distributed in a strong stripe in parasegment 6, but only low levels are seen in regions of the presumptive abdomen containing high levels of iab-4 transcript. No Ubx protein is detected at this early stage, possibly due to the time required to transcribe the entire ~80-kb locus (Ronshaugen, 2005).
During the rapid phase of germband elongation, Ubx protein becomes detectable in the abdomen. At the conclusion of germband elongation, the Ubx and iab-4 patterns are largely complementary in the dorsal ectoderm. During segmentation of the germband, the Ubx protein shows complex modulation in the dorsal ectoderm. There are three apparent levels of Ubx protein distribution in these regions: high, low, and none. An inverse correlation is noted between the levels of Ubx protein and the sites of iab-4 expression. The strongest expression of iab-4 occurs in regions having the lowest accumulation of Ubx protein, whereas intermediate and low levels coincide with sites of diminished Ubx accumulation; there is little or no iab-4 expression in those cells containing the highest levels of Ubx protein. These observations are consistent with the possibility that Ubx protein synthesis might be modulated by one or both iab-4 miRNAs. Direct support for this possibility stems from the analysis of a GFP-Ubx transgene containing the 3' UTR sequence from Ubx. This transgene displays slightly diminished expression in abdominal regions containing high levels of iab-4 transcripts (Ronshaugen, 2005).
Reference names in red indicate recommended papers.
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Brennecke, J., Hipfner, D. R., Stark, A., Russell, R. B., and Cohen, S. M. (2003). bantam encodes a developmentally regulated microRNA that controls cell proliferation and regulates the proapoptotic gene hid in Drosophila. Cell 113: 25-36. 12679032
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Ronshaugen, M., Biemar, F., Piel, J., Levine, M. and Lai, E. C. (2005)
Stark, A., Brennecke, J., Russell, R. B. and Cohen, S. M. (2003). Identification of Drosophila MicroRNA targets. PLoS Biol. 1: E60. 14691535
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date revised: 10 August 2006
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