The iab-4 noncoding RNA from the Drosophila bithorax complex is the substrate for a microRNA (miRNA). Gene conversion was used to delete the hairpin precursor of this miRNA; flies homozygous for this deletion are sterile. Surprisingly, this mutation complements with rearrangement breakpoint mutations that disrupt the iab-4 RNA but fails to complement with breaks mapping in the iab-5 through iab-7 regulatory regions. These breaks disrupt the iab-8 RNA, transcribed from the opposite strand. This iab-8 RNA also encodes a miRNA, detected on Northern blots, derived from the hairpin complementary to the iab-4 precursor hairpin. Ultrabithorax is a target of both miRNAs, although its repression is subtle in both cases (Bender, 2008).
A large number of microRNA (miRNA) clones prepared from Drosophila RNA have been characterized at a variety of developmental stages. Two of these clones matched sequences from the BX-C, mapping to the 3' end of a ncRNA discovered by Cumberledge (1990). This ncRNA was called the 'iab-4 RNA,' because it was thought to come from the iab-4 segmental domain of the BX-C, and the miRNAs were named miR-iab-4 5p (five prime) and miR-iab-4 3p (three prime). More recent mapping of segmental domains (Bender, 2000) has shown that the RNA actually lies in the iab-3 domain (regulating parasegment 8), and indeed, the ncRNA is expressed from parasegment 8 through parasegment 12 (Cumberledge, 1990). However, the iab-4 nomenclature is maintained here to avoid confusion with the designations in other studies. Two cDNA clones for the iab-4 RNA were described by Cumberledge (1990), with alternate 3' poly(A) sites separated by 304 base pairs (bp). The two miRNAs come from this region between these two poly(A) sites; both are presumably derived from a 70-base hairpin RNA precursor predicted from the sequence (Bender, 2008).
A recent study (Ronshaugen, 2005) suggested that the miR-iab-4 5p miRNA might be responsible for repression of Ubx in the abdominal segments where the miRNA is expressed. The conclusion was based on experiments in which miR-iab-4 5p was expressed at high levels in tissues, including the wing and haltere discs, where miR-iab-4 5p is not normally found. However, the pattern of UBX expression in PS8, where the miRNA is expressed, is very similar to the UBX pattern in PS7, which lacks the miRNA. The obvious repression of Ubx in both of these parasegments is clearly dependent on abd-A; any effect of miR-iab-4 5p must be subtle or redundant. Moreover, misexpression of miRNAs in other systems have been shown to give misleading effects. The function of miR-iab-4 5p can best be examined by mutating or deleting the miRNA (Bender, 2008).
Prior studies characterized a large number of P element insertions in the BX-C, including one called HCJ200, which maps only ~200 bp proximal to the miRNAs. This provided the opportunity to mutate the miRNAs by P-element-mediated gene conversion. Loss of the miRNAs derived from the iab-4 ncRNA causes no apparent morphological or behavioral phenotype, but the analysis revealed a functional miRNA derived from the opposite strand (Bender, 2008).
A 3.7-kb conversion donor fragment was constructed with a mutated version of the miRNA precursor sequence. The precursor sequence is symmetrically cut by the BstZ17I restriction endonuclease; this permitted the replacement of most of the precursor sequence with a double-stranded oligonucleotide-containing sites for the HindIII and I-SceI endonucleases. A plasmid with the donor sequence and a plasmid to supply P-element transposase were both injected into embryos with the HCJ200 (rosy+) P insertion. Offspring from the injected individuals were screened for loss of the HCJ200 P element (i.e., rosy-), and progeny from these exceptional flies were screened by PCR for a change in the size of the genomic DNA at the site of the insertion. One of 86 fertile injected animals gave the expected convertants. The conversion events were verified by sequencing the PCR product, and by whole-genome Southern blots. Genomic DNA was cut with HindIII, which cuts the oligonucleotide introduced in the conversion but does not cut the wild-type sequence within the 3.7-kb donor fragment. The sizes of the HindIII fragments on the Southern blot confirmed that the donor sequence was at the expected position in the BX-C and not at another genomic location (Bender, 2008).
Flies homozygous for the conversion chromosome (henceforth called 'δ') appeared normal. In particular, no evidence of segmental transformation was seen in mounted adult abdominal cuticles of either sex. However, both sexes were sterile. Females had ovaries with eggs of normal size, but only very rare individuals ever laid an egg, even after mating with wild-type males, and these rare eggs never hatched. Males had morphologically normal testes containing motile sperm. In single-fly tests for mating behavior, δ homozygous females mated with wild-type males as readily as their heterozygous siblings. The δ homozygous males showed normal courtship behavior toward wild-type females, except that they never completed copulation. The mutant males mounted the females, but they did not bend their abdomens quite far enough to mate. Thus, the sterility in both sexes appeared to be behavioral, due to a defect in the nerves or muscles required to lay eggs or to curl the abdomen (Bender, 2008).
The δ mutation was tested for complementation with rearrangement mutations in the BX-C, including several that should disrupt the iab-4 RNA transcript upstream of the position of the miRNA precursor. Surprisingly, breaks disrupting the iab-4 transcription unit complemented with δ -- i.e., trans-heterozygotes were fertile. Thus, the iab-4 RNA is not the precursor for any miRNA that is responsible for fertility. In contrast, rearrangements distal to the position of the miRNAs failed to complement, even with breaks >50 kb distal. Assuming that noncomplementing rearrangements are upstream in the precursor, one can deduce that the precursor is transcribed distal to proximal on the chromosome, and that the miRNA responsible for fertility comes from the opposite strand to those detected by Aravin (2003). Similarly, one would predict that the precursor transcript for the fertility miRNA spans at least the iab-7 through the iab-3 segmental domains (Bender, 2008).
Several studies have detected such a transcript (Sánchez-Herrero, 1989; Zhou, 1999; Bae, 2002; Bender, 2002), which is usually called the iab-8 ncRNA. It has been detected and mapped solely by in situ hybridization to RNA in embryos, although the complementation analysis now corroborates the in situ mapping, at least for the iab-4 through iab-7 region. Its start site is near Abd-B; it was detected by probes 4 kb proximal to the 3' end of the Abd-B transcripts. Zhou (1999) defined a potential promoter for the iab-8 transcript, which lies ~5 kb downstream from Abd-B, although the evidence did not preclude a start site further upstream. Bae (2002) detected the iab-8 RNA upstream of the Abd-B class A RNA start site (the 'BPP' probe, suggesting a more distal start site. However, hybridization to the Abd-B class B RNA in the ninth abdominal segment (PS14) could have been mistaken for the iab-8 RNA pattern. Moreover, the iab-8S10 breakpoint, just proximal to Abd-B, does complement the sterility phenotype of δ, and so the promoter for the iab-8 fertility function should be to the left of that break (Bender, 2008).
At the 3' end, the iab-8 RNA extends through abd-A. The iab-8 RNA in situ pattern was detected by a probe 5.5 kb proximal to the 3' end of the abd-A poly(A) site. Thus the transcription unit spans at least 120 kb. The iab-8 RNA has not been detected by probes in the bxd regulatory region, further proximal to abd-A (Bender, 2008).
The iab-8 transcript initiates at the cellular blastoderm stage (Zhou, 1999), as do most of the other embryonic ncRNAs (Akam, 1985; Cumberledge, 1990; Bae, 2002). However, it should take ~45 min to transcribe to the position of the miRNA precursor hairpin, assuming a transcription speed of ~1.3 kb/min. This would account for the developmental delay in the appearance of the RNA signal. The iab-8 RNA is located in the eighth abdominal segment and in more posterior segmental rudiments. In late embryos, the transcript persists in the posterior end of the ventral nerve chord (Bender, 2008).
Embryos homozygous for the δ mutation showed no apparent changes in the patterns of ABD-A and ABD-B, but there were subtle differences in the UBX pattern. UBX is expressed strongly and comprehensively in the cells of parasegment 6 (PS6, primarily the first abdominal segment). In the second abdominal segment (PS7), ABD-A appears and turns off UBX, especially in the more anterior cells of the parasegment. In the more posterior segments, the UBX staining pattern weakens progressively, and the ABD-A pattern becomes somewhat stronger. However, in δ embryos, the UBX pattern is nearly constant from PS7 through PS12. Thus, the progressive posterior decline in wild-type embryos appears not to be caused by ABD-A or ABD-B but rather by miR-iab-4 5p, whose expression shows a progressive posterior increase in PS8-12 (Bender, 2008).
In the eighth abdominal segment (PS13) of a wild-type embryo, UBX is almost completely absent in both the epidermis and the CNS. Homozygotes for δ show a partial derepression of UBX in the CNS in PS13. The derepression is similar in pattern and intensity to that seen in AbdB-/+ heterozygotes (data not shown). The repression of Ubx could be indirect; miR-iab-8 could be a positive regulator of Abd-B (and miR-iab-4 a positive regulator of abd-A). But all known targets of miRNAs are negatively regulated, and so it seems more likely that both miRNAs directly regulate Ubx (Bender, 2008).
It is possible that the effects of these two miRNAs are masked by functional redundancy with abd-A (for miR-iab-4) and Abd-B (for miR-iab-8). Embryos lacking ABD-A (but retaining miR-iab-4) show a dramatic derepression of UBX in the second through seventh abdominal segments. There does appear to be a slight decline in UBX levels in the more posterior segments, which could be attributed to miR-iab-4 repression. A complete analysis would include the UBX expression in embryos lacking both abd-A and miR-iab-4, but that will require construction of an abd-A, δ double mutant chromosome. In any case, miR-iab-4 repression of Ubx is subtle, even in the absence of ABD-A. Similarly, in an Abd-B homozygous mutant embryo (retaining miR-iab-8), UBX expression in the eighth abdominal segment closely resembles that in the seventh. Again, a δ, Abd-B double mutant chromosome would be useful for comparison, but again it is clear that the repression of Ubx by miR-iab-8 is still subtle in the absence of ABD-B. There is no reason to expect that Ubx is the only target of these miRNAs; perhaps other target genes will be discovered which the miRNAs repress more dramatically (Bender, 2008).
MiR-iab-8 is the first example of a functional product of a ncRNA in the BX-C. There are no other predicted miRNA precursor sequences in the iab-8 RNA or elsewhere in the BX-C (the Antennapedia complex includes miR-10), but there are many other ncRNAs. The possibility that they also include functional products now seems more likely (Bender, 2008).
Many microRNA loci exhibit compelling hairpin structures on both sense and antisense strands; however, the possibility that a miRNA gene might produce functional species from its antisense strand has not been examined. Antisense transcription of the Hox miRNA locus mir-iab-4 generates the novel pre-miRNA hairpin mir-iab-8, which is then processed into endogenous mature miRNAs. Sense and antisense iab-4/iab-8 miRNAs are functionally distinguished by their distinct domains of expression and targeting capabilities. miR-iab-8-5p, like miR-iab-4-5p, is also relevant to Hox gene regulation. Ectopic mir-iab-8 can strongly repress the Hox genes Ultrabithorax and abdominal-A via extensive arrays of conserved target sites, and can induce a dramatic homeotic transformation of halteres into wings. The antisense miRNA principle is generalizable: it has been shown that several other loci in both invertebrates and vertebrates are endogenously processed on their antisense strands into mature miRNAs with distinct seeds. These findings demonstrate that antisense transcription and processing contributes to the functional diversification of miRNA genes (Tyler, 2008).
These studies of BX-C miRNAs reveal two principal insights into miRNA regulatory biology. First, a new Hox cluster miRNA, mir-iab-8, was identified. Using gain-of-function assays it was shown that it can strongly inhibit Ubx and abd-A and generate homeotic phenotypic transformations. Indeed, the Hox-regulatory properties of mir-iab-8 are far more potent than those of mir-iab-4 (Ronshaugen, 2005), and correlate directly with the properties of its target sites in their 3' UTRs. Curiously, both BX-C miRNAs obey organizational and functional rules previously defined for the protein-encoding members of the BX-C. These regulatory RNAs exhibit colinearity, in that transcription of pri-mir-iab-8 initiates more distally on the chromosome and is expressed more posteriorly in the embryo relative to pri-mir-iab-4. They also exhibit posterior prevalence, in that both sense and antisense iab-4 miRNAs directly repress multiple homeotic genes located more anteriorly in the Hox cluster. In fact, the next most-anterior Hox gene Antp is a third likely endogenous iab-miRNA target that contains highly conserved target sites with t1A features for miR-iab-8-5p. In contrast, Abd-B contains no conserved sites for either iab-4 or iab-8 miRNAs in its long (>2 kb) 3' UTR. Therefore, BX-C miRNAs and homeobox genes are governed by the same regulatory logic (Tyler, 2008).
It is worth recalling that saturation mutagenesis screens of the BX-C revealed only three loci that are required for viability and exhibit homeotic defects, corresponding to the homeobox genes Ubx, abd-A, and Abd-B. In contrast, pioneering studies by Lewis (1978) considered rearrangements and dominant alleles suggested the existence of at least eight homeotic 'factors' in this region of the genome. Although many of these are now recognized as cis-regulatory elements that regulate Hox gene transcription, this work with BX-C miRNAs reveals two bona fide Hox regulators that are capable of inducing severe dominant homeotic transformations. The endogenous requirement for iab-4/iab-8 miRNAs appears to be subtle, possibly due to compensatory transcriptionally based regulatory mechanisms. Nevertheless, loss of function analysis corroborates that these miRNAs are required for normal expression of Hox targets in the nervous system and for normal development. These data emphasize that loss-of-function and gain-of-function genetics are complementary approaches to uncover important regulatory molecules (Tyler, 2008).
Antisense transcription and processing were uncovered in this study as a mechanism to generate new functional miRNAs. Bioinformatic analysis suggests that a large fraction of miRNA loci are theoretically competent to produce antisense miRNAs. Extant cloning efforts suggest that few miRNA loci actively produce large quantities of antisense miRNAs. Nevertheless, the sequencing effort reported in this study has revealed additional instances of putative antisense miRNAs. Although none of these was cloned more than twice, genetics demonstrates that rare miRNAs (e.g., lsy-6, expressed by a handful of neurons in a whole animal) can be critical components of regulatory networks and can have potent biological activities. Therefore, assessment of the biological relevance of the other antisense miRNA candidates awaits further study (Tyler, 2008).
In the case of the iab-4 locus, the regulatory diversity afforded by sense and antisense transcription of a single miRNA hairpin is manifested by altering the seed regions of their respective miRNA products and by deploying the sense and antisense pri-miRNA transcripts in distinct spatial domains. It might in fact be deleterious for a given locus to be simultaneously transcribed on both strands -- either because of transcriptional interference from colliding polymerase complexes, or because of the possibility to inadvertently generate dsRNA. Further analysis is needed to test the notion that it is favorable for sense/antisense miRNA pairs not to be expressed in the same cells. Overall, though, as animal genomes are quite extensively transcribed, and many miRNA genes adopt extensive hairpins on both strands, the potential for endogenous antisense processing of miRNA hairpins is theoretically quite broad. It is proposed that antisense transcription of other miRNA loci might generate novel small RNAs whose potentially beneficial regulatory activities are available for selection and stabilization by natural selection. This identification of several confident examples of antisense miRNAs, whose processing and/or targets have been conserved among diverse species, provides compelling support for this hypothesis (Tyler, 2008).
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).
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date revised: 15 July 2008
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