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The Drosophila Microrna Iab-4 Causes a Dominant Homeotic Transformation of Halteres to Wings

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The Drosophila Microrna Iab-4 Causes a Dominant Homeotic Transformation of Halteres to Wings Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press RESEARCH COMMUNICATION ample, miR-10 is located within the Drosophila Anten- The Drosophila microRNA napedia gene complex (ANT-C) between the Hox genes iab-4 causes a dominant Deformed and Sex combs reduced (Lagos-Quintana et al. 2001). The sequence and location of miR-10 are con- homeotic transformation of served in vertebrate Hox complexes (Tanzer et al. 2005). halteres to wings Sex combs reduced has been proposed as a direct miR-10 target in insects (Brennecke et al. 2005). A second group Matthew Ronshaugen,1 Frédéric Biemar,1 of Drosophila miRNAs map to a hairpin located at the 1 1,4 2,3 distal end of the iab-4 locus (Aravin et al. 2003), which Jessica Piel, Mike Levine, and Eric C. Lai resides between abd-A and Abd-B (Fig. 1A). miRNAs 1Department of Molecular and Cell Biology, Division of were cloned from both arms of this hairpin and are Genetics, Center for Integrative Genomics, University of termed iab-4–5p and iab-4–3p (Aravin et al. 2003). miR– California, Berkeley, California 94720, USA; 2Department of iab-4–5p was recently predicted to regulate Ubx activity Developmental Biology, Memorial Sloan-Kettering Cancer (Stark et al. 2003; Grun et al. 2005). Although vertebrates Center, New York, New York 10021, USA lack an iab-4 ortholog, as defined by sequence identity, a different miRNA, miR-196, resides at an analogous po- The Drosophila Bithorax Complex encodes three well- sition adjacent to the posterior-most HOX 9–13 paralogs. characterized homeodomain proteins that direct seg- Tissue culture assays, in vivo cleavage products, and ment identity, as well as several noncoding RNAs of un- transgenic lacZ “sensors” indicate that miR-196 inhibits Hoxb8 activity (Mansfield et al. 2004; Yekta et al. 2004). known function. Here, we analyze the iab-4 locus, Despite these provocative target relationships, no phe- which produces the microRNAs iab-4–5p and iab-4–3p. notypes have been associated with any Hox miRNA. iab-4 is analogous to miR-196 in vertebrate Hox clusters. miRNAs are short, 21–24-nt RNAs that attenuate pro- Previous studies demonstrate that miR-196 interacts tein synthesis by binding complementary sites in target with the Hoxb8 3؅ untranslated region. Evidence is pre- mRNAs (Lai 2003; Bartel 2004). An unexpectedly modest sented that miR–iab-4–5p directly inhibits Ubx activity amount of base-pairing appears to underlie target recog- in vivo. Ectopic expression of mir–iab-4–5p attenuates nition. Experimental and computational studies have endogenous Ubx protein accumulation and induces a converged on the principle of “seed-pairing,” whereby ∼7 Ј classical homeotic mutant phenotype: the transforma- continuous Watson-Crick base pairs at the 5 -end of the tion of halteres into wings. These findings provide the miRNA mediate target recognition (Lai 2002; Brennecke et al. 2005; Lewis et al. 2005). The limited sequence re- first evidence for a noncoding homeotic gene and raise quirement for miRNA–mRNA interactions has fueled the possibility that other such genes occur within the current proposals that a third or more of all mRNAs may Bithorax complex. We also discuss the regulation of mir– be regulated by miRNAs (Lewis et al. 2005; Xie et al. iab-4 expression during development. 2005). As tallies of miRNA loci continue to grow (with Received September 6, 2005; revised version accepted October current estimates for humans ranging from 800 to 1000) 31, 2005. (Bentwich et al. 2005; Berezikov et al. 2005), the network of possible miRNA:target interactions will expand. Only a small number of miRNA:target interactions Classical genetic studies suggest that the Bithorax Com- have been studied in vivo. Here we present evidence that plex (BX-C) contains as many as nine homeotic genes iab-4 microRNAs selectively attenuate Ubx activity in (Lewis 1978). However, only three encode Hox proteins, vivo. The Ubx 3Ј untranslated region (3Ј UTR) contains Ultrabithorax (Ubx), abdominal-A (abd-A), and Ab- predicted target sites for miR–iab-4–5p and expression of dominal-B (Abd-B) (Martin et al. 1995). The bulk of ge- a GFP-Ubx-3Ј UTR “sensor” transgene is repressed by nomic DNA comprising the BX-C is thought to function ectopic expression of a mir–iab-4 minigene. This mini- as cis-regulatory DNA that controls the timing and site of gene also reduces Ubx protein levels in haltere imaginal Hox expression (Sanchez-Herrero et al. 1985). Neverthe- discs, thereby inducing a classical homeotic transforma- less, it has been known for more than 15 years that in- tion of halteres into wings. Taken together, these results tergenic regions of the BX-C are extensively transcribed suggest that the iab-4 transcription unit encodes an au- (Cumberledge et al. 1990; Bae et al. 2002; Drewell et al. thentic homeotic regulatory gene. We suggest that addi- 2002). The possible functional activities of the noncod- tional noncoding RNAs correspond to “missing” homeo- ing RNAs have received little attention, despite the fact tic genes in the Bithorax complex, and discuss novel that these transcripts, including iab-4, are expressed in mechanisms of iab-4 regulation during development. restricted domains along the anterior–posterior axis, like conventional Hox genes (Cumberledge et al. 1990). Results and Discussion Hox gene clusters contain conserved miRNAs. For ex- Complementary patterns of iab-4 and Ubx expression Among the sequenced Drosophilids, there are only a few [Keywords: microRNA; iab-4; Ultrabithorax; homeotic gene] highly conserved sequences in the 120-kb region sepa- Corresponding authors. rating abd-A and Abd-B in the BX-C (Fig. 1A). Three are 3 E-MAIL [email protected]; FAX (212) 717-3604. located in regions that flank known insulator elements 4E-MAIL [email protected]; FAX (510) 643-5780. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ (Karch et al. 1994; Zhou et al. 1996; Barges et al. 2000). A gad.1372505. fourth conserved region is located within the 3Ј region of the GENES & DEVELOPMENT 19:2947–2952 © 2005 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/05; www.genesdev.org 2947 Downloaded from genesdev.cshlp.org on October 6, 2021 - Published by Cold Spring Harbor Laboratory Press Ronshaugen et al. used to determine the relative expression patterns of iab-4 RNA and Ubx RNA/protein accumulation during embryonic development (Fig. 1C–E). The iab-4 primary transcript is strongly expressed in the presumptive abdo- men, mainly in the progenitors of the second (A2) through seventh (A7) segments (see Cumberledge et al. 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 lev- els of iab-4 transcript. No Ubx protein is detected at this early stage, possibly due to the time required to tran- scribe the entire ∼80-kb locus (Shermoen and O’Farrell 1991). During the rapid phase of germband elongation, Ubx protein becomes detectable in the abdomen (Fig. 1D). At the conclusion of germband elongation, the Ubx and iab-4 patterns are largely complementary in the dorsal ectoderm (Fig. 1E). During segmentation of the germ- Figure 1. Structure, conservation, and expression of the iab-4 miRNA. (A) A 120-kb interval of the D. melanogaster Bithorax band, the Ubx protein shows complex modulation in the Complex that includes the homeobox genes abd-A and Abd-B, the dorsal ectoderm (Fig. 1F–H). There are three apparent noncoding RNA gene iab-4, and three transcribed boundary ele- levels of Ubx protein distribution in these regions: high, ments (MCP, Fab-7, and Fab-8). Shown are VISTA plots depicting low, and none (Fig. 1F). We note an inverse correlation highly conserved sequences in the distantly related Drosophilid between the levels of Ubx protein and the sites of iab-4 Drosophila mojaviensis (regions of >80% identity in a window of 50 expression (Fig. 1G,H). The strongest expression of iab-4 nt) and the honeybee A. mellifera (regions of >80% identity in a window of 150 nt). Note that all of these conserved regions are occurs in regions having the lowest accumulation of Ubx associated with transcribed noncoding elements. Depicted at higher protein (Fig. 1F–H, arrows), whereas intermediate and magnification are two iab-4 splice forms, an ∼2.1-kb transcript con- low levels coincide with sites of diminished Ubx accu- taining the iab-4 miRNA hairpin and a shorter transcript that ter- mulation (Fig. 1F–H, asterisks); there is little or no iab-4 minates just 5Ј of the miRNAs. The two iab-4 probes used in this expression in those cells containing the highest levels of study (iab-4 probe1 and iab-4 probe2) are shown as black bars below Ubx protein (Fig. 1F–H, arrowheads). These observations the schematics in A.(B) Perfect conservation of the iab-4–5p and iab-4–3p miRNAs in diverse arthropods: D. melanogaster (D. mel), are consistent with the possibility that Ubx protein syn- D. pseudoobscura (D. pse), Bombyx mori (B. mor), A. mellifera (A. thesis might be modulated by one or both iab-4 miRNAs. mel), A. aegypti (A. egy), A. gambiae (A. gam), and T. castaneum (T. Direct support for this possibility stems from the analy- cas). (C–E) Time course of iab-4 (red) and Ubx (green) nascent tran- sis of a GFP-Ubx transgene containing the 3Ј UTR se- scription: stage 5 (C), stage 8 (D), stage 11 (E). Nascent iab-4 tran- quence from Ubx.
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