Gene name - iab-4
Synonyms - nuclear untranslated RNA gene iab-4
Cytological map position - 89E2
Function - translational repression
Symbol - iab-4
FlyBase ID: FBgn0020546
Genetic map position - 3-
Classification - untranslated RNA gene
Cellular location - nucleus
The Drosophila Bithorax Complex encodes three well-characterized homeodomain proteins that direct segment identity, as well as several noncoding RNAs of unknown function. This study analyzes the iab-4 locus, which produces the microRNAs iab-4-5p and iab-4-3p. iab-4 is analogous to miR-196 in vertebrate Hox clusters. Previous studies demonstrated that miR-196 interacts with the Hoxb8 3' untranslated region. Evidence is presented that miR-iab-4-5p directly inhibits Ubx activity in vivo. Ectopic expression of mir-iab-4-5p attenuates endogenous Ubx protein accumulation and induces a classical homeotic mutant phenotype: the transformation of halteres into wings. These findings provide the first evidence for a noncoding homeotic gene and raise the possibility that other such genes occur within the Bithorax complex (Ronshaugen, 2005).
Classical genetic studies suggest that the Bithorax Complex (BX-C) contains as many as nine homeotic genes (Lewis 1978). However, only three encode Hox proteins: Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B). The bulk of genomic DNA comprising the BX-C is thought to function as cis-regulatory DNA which controls the timing and site of Hox expression. Nevertheless, it has been known for more than 15 years that intergenic regions of the BX-C are extensively transcribed (Cumberledge, 1990; Bae, 2002; Drewell, 2002). The possible functional activities of the noncoding RNAs have received little attention, despite the fact that these transcripts, including iab-4 (Cumberledge, 1990), are expressed in restricted domains along the anterior-posterior axis, like conventional Hox genes (Ronshaugen, 2005).
Hox gene clusters contain conserved miRNAs. For example, miR-10 is located within the Drosophila Antennapedia gene complex (ANT-C) between the Hox genes Deformed and Sex combs reduced (Lagos-Quintana, 2001). The sequence and location of miR-10 are conserved in vertebrate Hox complexes (Tanzer, 2005). Sex combs reduced has been proposed as a direct miR-10 target in insects (Brennecke, 2005). A second group of Drosophila miRNAs map to a hairpin located at the distal end of the iab-4 locus (Aravin, 2003), which resides between abd-A and Abd-B (). miRNAs were cloned from both arms of this hairpin and are termed iab-4-5p and iab-4-3p (Aravin, 2003). miR-iab-4-5p was recently predicted to regulate Ubx activity (Stark, 2003; Grun, 2005). Although vertebrates lack an iab-4 ortholog, as defined by sequence identity, a different miRNA, miR-196, resides at an analogous position adjacent to the posterior-most HOX 9-13 paralogs. Tissue culture assays, in vivo cleavage products, and transgenic lacZ 'sensors' indicate that miR-196 inhibits Hoxb8 activity (Mansfield, 2004; Yekta, 2004). Despite these provocative target relationships, no phenotypes have been associated with any Hox miRNA (Ronshaugen, 2005).
miRNAs are short, 21-24-nt RNAs that attenuate protein synthesis by binding complementary sites in target mRNAs. An unexpectedly modest amount of base-pairing appears to underlie target recognition. Experimental and computational studies have converged on the principle of 'seed-pairing,' whereby ~7 continuous Watson-Crick base pairs at the 5'-end of the miRNA mediate target recognition. The limited sequence requirement for miRNA-mRNA interactions has fueled current proposals that a third or more of all mRNAs may be regulated by miRNAs. As tallies of miRNA loci continue to grow (with current estimates for humans ranging from 800 to 1000), the network of possible miRNA:target interactions will expand (Ronshaugen, 2005).
Only a small number of miRNA:target interactions have been studied in vivo. This study presents evidence that iab-4 microRNAs selectively attenuate Ubx activity in vivo. The Ubx 3' untranslated region (3' UTR) contains predicted target sites for miR-iab-4-5p and expression of a GFP-Ubx-3' UTR 'sensor' transgene is repressed by ectopic expression of a mir-iab-4 minigene. This minigene also reduces Ubx protein levels in haltere imaginal discs, thereby inducing a classical homeotic transformation of halteres into wings. Taken together, these results suggest that the iab-4 transcription unit encodes an authentic homeotic regulatory gene. It is suggested that additional noncoding RNAs correspond to 'missing' homeotic genes in the Bithorax complex, and novel mechanisms of iab-4 regulation during development are discussed (Ronshaugen, 2005).
The detailed analysis of intergenic transcripts in the abd-A/Abd-B interval suggests a 'strand exclusion' model for iab-4 regulation. The iab-4 locus is unusual in that both strands are transcribed (Bae, 2002). However, each strand displays a distinct pattern of expression. The strand producing the iab-4 pri-miRNA is broadly expressed in the A2-A7 region of the germband, while the other strand is expressed in A8 and A9. Double RNA FISH assays suggest that the miR strand is initially expressed in A2-A8, but expression is lost in A8 as transcription of the other strand progresses from the iab-8 domain. Perhaps transcription from one strand diminishes transcription from the other. Although the detailed mechanism may be different, these observations are evocative of the mutually exclusive expression of Xist and Tsix RNAs on mammalian X chromosomes. Additional target predictions for miR-iab-4 imply that exclusion of iab-4 expression from A8 might be important for stable accumulation of other potential iab-4 target mRNAs, such as Abd-B (Enright, 2003; Stark, 2003; Ronshaugen, 2005 and references therein).
Traditionally, recessive loss-of-function mutations are used to assess the in vivo activities of patterning genes. The principal argument for iab-4:Ubx interactions in development rests with the analysis of dominant gain-of-function phenotypes arising from the misexpression of miR-iab-4 in the haltere imaginal discs. The specificity of the resulting haltere-to-wing homeotic transformation correlates with reduced levels of Ubx protein accumulation specifically in the regions where miR-iab-4 products are misexpressed. It is likely that there is redundancy in the transcriptional repression of Ubx by Abd-A and Abd-B products, and the inhibition of Ubx protein synthesis by iab-4 miRNAs. Indeed, noncoding genes in the BX-C were mainly identified by dominant mutations such as chromosomal rearrangements. Therefore, misexpression assays may prove effective in analyzing the function of other Hox noncoding genes (Ronshaugen, 2005).
This study provides evidence that iab-4 encodes a novel homeotic regulatory activity, which functions, at least in part, by producing miRNAs inhibiting Ubx. iab-4 miRNAs may regulate additional target mRNAs. For example, computational analyses identify homothorax as another potential target of interest (Grun, 2005). Homothorax works in parallel with the Hox cofactor Extradenticle and various Hox proteins to control the patterning of legs and antennae. It is also possible that downstream transcriptional targets of Ubx ('realizators') might be modulated by iab-4 miRNAs. Additional noncoding RNAs in the BX-C, such as cbx, pbx and bxd, might also possess homeotic regulatory activities and account for the remaining genes identified within the sorrounding region (Ronshaugen, 2005).
MicroRNAs (miRNAs) are ~22-nucleotide RNAs that are processed from characteristic precursor hairpins and pair to sites in messages of protein-coding genes to direct post-transcriptional repression. The miRNA iab-4 locus in the Drosophila Hox cluster is transcribed convergently from both DNA strands, giving rise to two distinct functional miRNAs. Both sense and antisense miRNA products target neighboring Hox genes via highly conserved sites, leading to homeotic transformations when ectopically expressed. Sense/antisense miRNAs are also present in the mouse and antisense transcripts are found close to many miRNAs in both flies and mammals, suggesting that additional sense/antisense pairs exist (Stark, 2008).
Hox genes are highly conserved homeobox-containing transcription factors crucial for development in animals. Genetic analyses have identified them as determinants of segmental identity that specify morphological diversity along the anteroposterior body axis. A striking conserved feature of Hox complexes is the spatial colinearity between Hox gene transcription in the embryo and the order of the genes along the chromosome. Hox clusters also give rise to a variety of noncoding transcripts, including microRNAs (miRNAs) mir-10 and mir-iab-4/mir-196, which derive from analogous positions in Hox clusters in flies and vertebrates (Yekta, 2004). miRNAs are ~22-nucleotide (nt) RNAs that regulate gene expression post-transcriptionally. They are transcribed as longer precursors and processed from characteristic pre-miRNA hairpins. In particular, Hox miRNAs have been shown to regulate Hox protein-coding genes by mRNA cleavage and inhibition of translation, thereby contributing to the extensive regulatory connections within Hox clusters (Mansfield, 2004; Yekta, 2004; Hornstein, 2005; Ronshaugen, 2005). Several Hox transcripts overlap on opposite strands, providing evidence of extensive antisense transcription, including antisense transcripts for mir-iab-4 in flies (Bae, 2002) and its mammalian equivalent mir-196 (Mainguy, 2007). However, the function of these transcripts has been elusive. This study shows that the iab4 locus in Drosophila produces miRNAs from opposite DNA strands that can regulate neighboring Hox genes via highly conserved sites. Evidence is provided that such sense/antisense miRNA pairs are likely employed in other contexts and a wide range of species (Stark, 2008).
Examination of the antisense transcript that overlaps Drosophila mir-iab-4 revealed that the reverse complement of the mir-iab-4 hairpin folds into a hairpin reminiscent of miRNA precursors. Moreover, 17 sequencing reads from small RNA libraries of Drosophila testes and ovaries mapped uniquely to one arm of the iab-4 antisense hairpin. All reads were aligned at their 5' end, suggesting that the mir-iab-4 antisense hairpin is processed into a single mature miRNA in vivo, which is referred to as miR-iab-4AS. For comparison, six reads were found consistent with the known miR-iab-4-5p (or miR-iab-4 for short) and one read was foudn for its star sequence (miR-iab-4-3p). Interestingly, the relative abundance of mature miRNAs and star sequences for mir-iab-4AS (17:0) and mir-iab-4 (6:1) reflects the thermodynamic asymmetry of the predicted miRNA/miRNA* duplexes (Khvorova, 2003; Schwarz, 2003). Because they derived from complementary near palindromes, miR-iab-4 and miR-iab-4AS had high sequence similarity, only differing in four positions at the 3' region. However, they differed in their 5' ends, which largely determine miRNA target spectra (Brennecke, 2005; Lewis, 2005): miR-iab-4AS was shifted by 2 nt, suggesting targeting properties distinct from those of miR-iab-4 and other known Drosophila miRNAs (Stark, 2008).
Robust transcription of mir-iab-4 sense and antisense precursors was confirmed by in situ hybridization to Drosophila embryos. Both transcripts were detected in abdominal segments in the posterior part of the embryo, but intriguingly in nonoverlapping domains. As described previously (Bae, 2002; Ronshaugen, 2005), mir-iab-4 sense was expressed highly in abdominal segments A5-A7, showing modulation in levels within the segments: abdominal-A (abd-A)-expressing cells appeared to have more mir-iab-4, whereas Ultrabithorax (Ubx)-positive cells appeared to have little or none (this study; Ronshaugen, 2005). In contrast, mir-iab-4AS transcription was detected in the segments A8 and A9, where Abdominal-B (Abd-B) is known to be expressed (this study; Yoder, 2006). Primary transcripts for mir-iab-4 and mir-iab-4AS were also detected by strand-specific RT-PCR in larvae, pupae, and male and female adult flies, suggesting that both miRNAs are expressed throughout fly development (Stark, 2008).
To assess the possible biological roles of the two iab-4 miRNAs, fly genes were examined for potential target sites by searching for conserved matches to the seed region of the miRNAs (Lewis, 2005). Highly conserved target sites were found for miR-iab-4AS in the 3' untranslated regions (UTRs) of several Hox genes that are proximal to the iab-4 locus and are expressed in the neighboring more anterior embryonic segments: abd-A, Ubx, and Antennapedia (Antp) have four, five, and two seed sites, respectively, most of which are conserved across 12 Drosophila species that diverged 40 million years ago. More than two highly conserved sites for one miRNA is exceptional for fly 3' UTRs, placing these messages among the most confidently predicted miRNA targets and suggesting that they might be particularly responsive to the presence of the miRNA. The strong predicted targeting of proximal Hox genes was reminiscent of previously characterized miR-iab-4 targeting of Ubx in flies and miR-196 targeting of HoxB8 in vertebrates (Mansfield, 2004; Yekta, 2004; Hornstein, 2005; Ronshaugen, 2005; Stark, 2008 and references therein).
To test whether miR-iab4AS is functional and can directly target abd-A and Ubx, Luciferase reporters were constructed carrying the corresponding wild-type 3' UTRs and control 3' UTRs in which each seed site was disrupted by point substitutions. mir-iab-4AS potently repressed reporter activity for abd-A and Ubx. This repression was specific to the miR-iab-4AS seed sites; expression of the control reporters with mutated sites was not affected. Tested were perform to see whether mir-iab-4AS reduced expression of a Luciferase reporter with the Abd-B 3' UTR, which has no seed sites. As expected, mir-iab-4AS expression did not affect reporter activity, consistent with a model where miRNAs do not target genes that are coexpressed at high levels. In addition to demonstrating specific repression dependent on the predicted target sites, these assays confirmed the processing of the mir-iab-4AS hairpin into a functional mature miRNA (Stark, 2008).
If miR-iab-4AS were able to potently down-regulate Ubx in the fly, its misexpression should result in a Ubx loss-of-function phenotype, a line of reasoning that has often been used to study the functions and regulatory relationships of Hox genes. Ubx is expressed throughout the haltere imaginal disc, where it represses wing-specific genes and specifies haltere identity. When mir-iab-4AS was expressed in the haltere imaginal disc under bx-Gal4 control, a clear homeotic transformation of halteres to wings was observed. The halteres developed sense organs characteristic of the wing margin and their size increased severalfold, features typical of transformation to wing. Consistent with the increased number of miR-iab4AS target sites, the transformation was stronger than that reported for expression of iab-4 (Ronshaugen, 2005), for which changes in morphology were confirmed wing-like growth was not found (Stark, 2008).
It is concluded that both strands of the iab-4 locus are expressed in nonoverlapping embryonic domains and that each transcript produces a functional miRNA in vivo. In particular, the novel mir-iab-4AS is able to strongly down-regulate neighboring Hox genes. Interestingly, vertebrate mir-196, which lies at an analogous position in the vertebrate Hox clusters, is transcribed in the same direction as mir-iab-4AS and most other Hox genes, and targets homologs of both abd-A and Ubx (Mansfield, 2004; Yekta, 2004; Hornstein, 2005). With its shared transcriptional orientation and homologous targets, mir-iab-4AS appears to be the functional equivalent of mir-196 (Stark, 2008).
The expression patterns and regulatory connections between Hox genes and the two iab-4 miRNAs show an intriguing pattern in which the miRNAs appear to reinforce Hox gene-mediated transcriptional regulation. In particular, miR-iab-4AS would reinforce the posterior expression boundary of abd-A, Ubx, and Antp, supporting their transcriptional repression by Abd-B. mir-iab-4 appears to support abd-A- and Abd-B-mediated repression of Ubx, reinforcing the abd-A/Ubx expression domains and the posterior boundary of Ubx expression. Furthermore, both iab-4 miRNAs have conserved target sites in Antp, which is also repressed by Abd-B, abd-A, and Ubx. The iab-4 miRNAs thus appear to support the established regulatory hierarchy among Hox transcription factors, which exhibits 'posterior prevalence,' in that more posterior Hox genes repress more anterior ones and are dominant in specifying segment identity. Interestingly, Abd-B and mir-iab-4AS are expressed in the same segments, and the majority of cis-regulatory elements controlling Abd-B expression are located 3' of Abd-B. This places them near the inferred transcription start of mir-iab-4AS, where they potentially direct the coexpression of these genes. Similarly, abd-A and mir-iab-4 may be coregulated as both are transcribed divergently, potentially under the control of shared upstream elements (Stark, 2008).
These data demonstrate the transcription and processing of sense and antisense mir-iab-4 into functional miRNAs with highly conserved functional target sites in neighboring Hox genes. In an accompanying study (Bender 2008), genetic and molecular analyses in mir-iab-4 mutant Drosophila revealed that the proposed regulation of Ubx by both sense and antisense miRNAs occurs under physiological conditions and, in particular, the regulation by miR-iab-4AS is required for normal development. These lines of evidence establish miR-iab-4AS as a novel Hox gene, being expressed from within the Hox cluster and regulating Hox genes during development (Stark, 2008).
The genomic arrangement of two miRNAs that are expressed from the same locus but on different strands might provide a simple and efficient means to create nonoverlapping miRNA expression domains. Such sense/antisense miRNAs could restrict each other's transcription, either by direct transcriptional interference, as shown for overlapping convergently transcribed genes (Shearwin, 2005; Hongay, 2006), or post-transcriptionally, possibly via RNA-RNA duplexes formed by the complementary transcripts. Sense/antisense miRNAs would usually differ at their 5' ends and thereby target distinct sets of genes, which might help define and establish sharp boundaries between expression domains. Coupled with feedback loops or coregulation of miRNAs and genes in cis or trans, this arrangement could provide a powerful regulatory switch. The iab-4 miRNAs might be a special case of tight regulatory integration in which miRNAs and proximal genes appear coregulated transcriptionally in cis and repress each other both transcriptionally and post-transcriptionally (Stark, 2008).
It is perhaps surprising that no antisense miRNA had been found previously, even though, for example, the intriguing expression pattern of the iab-4 transcripts had been reported nearly two decades ago (Cumberledge, 1990; Bae, 2002), and iab-4 lies in one of the most extensively studied regions of the Drosophila genome. The frequent occurrence of antisense transcripts (Yelin, 2003; Katayama, 2005) suggests that more antisense miRNAs might exist. Indeed, up to 13% of known Drosophila , 20% of mouse, and 31% of human miRNAs are located in introns of host genes transcribed on the opposite strand or are within 50 nt of antisense ESTs or cDNAs. These include an antisense transcript overlapping human mir-196 (see also Mainguy, 2007). However, because of the contribution of noncanonical base pairs, particularly G:U pairs that become less favorable A:C in the antisense strand, many miRNA antisense transcripts will not fold into hairpin structures suitable for miRNA biogenesis, which explains the propensity of miRNA gene predictions to identify the correct strand. Nonetheless, in a recent prediction effort, 22 sequences reverse-complementary to known Drosophila miRNAs showed scores seemingly compatible with miRNA processing. Deep sequencing of small RNA libraries from Drosophila confirmed the processing of small RNAs from four of these high-scoring antisense candidates (Ruby, 2007), and the ovary/testes libraries used here showed antisense reads for an additional Drosophila miRNA (mir-312). In addition, using high-throughput sequencing of small RNA libraries from mice, sequencing reads were found that uniquely matched the mouse genome in loci antisense to 10 annotated mouse miRNAs. Eight of the inferred antisense miRNAs were supported by multiple independent reads, and two of them had reads from both the mature miRNA and the star sequence. These results suggest that sense/antisense miRNAs could be more generally employed in diverse contexts and in species as divergent as flies and mammals (Stark, 2008).
The infra-abdominal (iab) regions of the biothorax complex are thought to cis-regulate expression of the abdominal-A (abd-A) and Abdominal-B (Abd-B) transcripts. These large cis-regulatory regions are also actively transcribed. A detailed analysis is presented of the transcription products of the iab-4 region. During early embryogenesis a 6.8-kilobase (kb) RNA is transcribed and processed to yield 1.7- and 2.0-kb poly(A)+ RNAs. These RNAs are expressed from the time of cellular blastoderm formation to germ-band shortening and are localized in parasegments 8-14 (the posterior second through the anterior ninth abdominal segments). Sequence analysis of the two RNAs suggests that they may not be translated. Some possible functions of the 1.7/2.0-kb iab RNAs and the significance of their similarity to the bithoraxoid (bxd) RNAs and other transcripts from the iab regions of the abd-A and Abd-B domains are discussed (Cumberledge, 1990).
Among the sequenced Drosophilids, there are only a few highly conserved sequences in the 120-kb region separating abd-A and Abd-B in the BX-C. Three are located in regions that flank known insulator elements. A fourth conserved region is located within the 3' region of the iab-4 transcription unit (Cumberledge, 1990). The iab-4 locus contains regulatory DNAs that control abd-A expression (Karch, 1990). It also produces two spliced, polyadenylated transcripts that differ by the presence or absence of the highly conserved 3' sequences. This region contains a single ~100-nucleotide (nt) pre-miRNA hairpin structure that encodes two stable miRNAs: iab-4-5p and iab-4-3p (Aravin, 2003; Ronshaugen, 2005).
The pre-mir-iab-4 hairpin is conserved both in sequence and genomic location in the mosquitoes Anopheles gambiae and Aedes aegypti, the honeybee Apis mellifera, and the flour beetle Tribolium castaneum, species that last shared a common ancestor roughly 400 million years ago. In fact, it is by far the best conserved sequence in the abd-A/Abd-B interval among Drosophilid and non-Drosophilid genomes. It is notable that the sequences corresponding to the mature iab-4-5p and iab-4-3p miRNAs (each forming one arm of the hairpin) are perfectly conserved among this broad spectrum of insects (Ronshaugen, 2005).
date revised: 10 August 2006
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