Ultrabithorax

Posttranscriptional regulation

Ultrabithorax and Antennapedia 5' untranslated regions promote developmentally regulated internal translation initiation. In principle, mRNAs that contain unusually long leader sequences with multiple upstream reading frames (URFs) are good candidates for initiating transcription via a cap-independent internal ribosome binding mechanism. The 5' untranslated regions (UTRs) of the Drosophila Ubx and Antp genes were tested for their ability to promote cap-independent translation initiation. The Ubx and the Antp 5' UTR were inserted between the CAT and lacZ coding sequences in a dicistronic gene and tested for internal ribosome entry site (IRES) activity in transgenic Drosophila. Predicted full-length dicistronic mRNAs were present. High CAT activity is expressed from the first cistron from all of the dicistronic constructs introduced into the fly genome. The dicistronic transgenic strains bearing the Ubx and Antp IRES elements express significant levels of beta-galactosidase (betaGAL) from the second cistron whereas little or no betaGAL is expressed in the controls lacking the IRESs. In situ analysis of betaGAL expression in the transgenic strains indicates that expression of the second cistron is spatially and temporally regulated. Although the developmental patterns of expression directed by the Antp and Ubx IRESs overlap, they exhibit several differences indicating that these IRESs are not functionally equivalent (Ye, 1997).

Alternative splicing of pre-mRNAs is a versatile regulatory mechanism that can achieve quantitative control of gene expression and functional diversification of gene products. Much progress has been made toward understanding the basic splicing reaction and recognizing exon/intron boundaries, but the mechanisms that regulate alternative splicing are only beginning to be elucidated. Recognition of the 5' splice site by U1 snRNP and of the branchpoint near the 3' splice site by U2 snRNP auxiliary factor (U2AF) are two critical early steps that are regulated in cell- or stage-specific alternative splicing. The picture emerging from biochemical and genetic studies is that splice site selection results from the combined action of conserved consensus sequences that base-pair with the U snRNAs together with protein-protein and protein-RNA interactions that stabilize snRNP binding and mediate bridging interactions between snRNPs at the 5' and 3' splice sites. These interactions involve a growing list of non-snRNP factors, some of which may be responsible for developmental regulation of splice site selection (Burnette, 1999 and references).

Members of the SR family of RNA-binding proteins are required for multiple steps of the splicing reaction in vitro and their concentration can influence splice site competition both in vitro and in overexpression assays using cultured cells. SR proteins are required for the activity of at least some splicing enhancers that stimulate the use of weak 5' or 3' splice sites, and there is evidence for distinct specificities in these interactions. Members of the hnRNP A/B family of RNA-binding proteins also influence splice site selection in a concentration-dependent manner in vitro and when overexpressed in cultured cells. In these assays the hnRNP RNA binding proteins can antagonize the action of SR proteins. These observations have suggested that SR proteins and hnRNP A/B proteins function in vivo as concentration-dependent regulators of alternative splicing. Another possibility is that members of these families serve as cofactors or targets for the actual regulators. Particular SR proteins have been proposed to interact with developmentally specific factors to promote regulation of splicing (Burnette, 1999 and references).

Although a framework of hypotheses is evolving, little is known about regulators of alternative splicing and how they function in vivo. Notable exceptions are Sex lethal and Transformer, proteins that control alternative splicing decisions during sex determination in Drosophila. Because few developmentally specific regulators of alternative splicing have been identified, it is possible that many -- if not most -- alternative splicing decisions are regulated by relatively subtle variations in the levels of general, widely distributed factors, perhaps acting cooperatively or antagonistically as proposed for SR and hnRNP A/B proteins. This is consistent with much correlative evidence and many in vitro observations, but conclusive proof that either type of protein normally regulates an alternative splicing decision in vivo has yet to be obtained. Although null alleles of the Drosophila SR protein gene B52 (homolog of human SRp55) show it to be essential for viability, examination of multiple constitutively and alternatively spliced RNAs has failed to reveal any alterations of splicing even in the absence of detectable protein (Burnette, 1999 and references).

The Ultrabithorax (Ubx) was used as a model for regulation of alternative splicing in large and complex transcription units. The six alternative Ubx mRNAs share large protein-coding 5' and 3' exons but differ in the pattern of incorporation of three elements: B is located between two alternative donor sites at the end of the first common exon, whereas mI and mII are internal cassette exons. Within the central nervous system (CNS), different neurons express distinct ratios of Ubx isoforms. The complex and quantitative nature of this regulation is unlike that of other well-studied model systems in Drosophila (e.g., sex-specific splicing in the sex determination hierarchy or germ line-specific splicing of P-element transcripts) but resembles that of many other genes in vertebrates and invertebrates. It seems most likely that this type of alternative splicing is controlled not by highly tissue- and gene-specific splicing regulators but by developmental variations in the concentration or activity of broadly distributed multifunctional factors that may act combinatorially. Hence, Ubx should be a valuable model where genetic approaches can be used to dissect this type of regulation (Burnette, 1999 and references).

Strong reductions of function for the postulated type of regulatory factors would probably cause lethal phenotypes that would be uninterpretable in terms of their effects on Ubx splicing. However, the Ubx splicing pattern should be sensitive to partial reductions in the concentration or activity of these regulatory factors. This may also be true for factors that play important accessory roles in regulation as targets or as constitutively expressed components of regulatory complexes. Two approaches were used to identify such factors. (1) First, a test was carried out to see if the Ubx alternative splicing pattern is altered in heterozygotes for strong loss-of-function mutations. Such mutations are found in a set of genes implicated in the control of alternative splicing in Sxl and P-element RNAs. (2) To identify the location of additional genes involved in regulation of Ubx splicing, a large collection of deficiencies was tested for dominant enhancement of the haploinsufficient Ubx haltere phenotype; it was then asked whether the Ubx splicing pattern is altered in heterozygotes for the interacting deficiencies, and the phenotypic interaction and effect on splicing was traced to specific genes when mutations existed in reasonable candidates. Inclusion of the cassette exons in Ubx mRNAs is reduced strongly in heterozygotes for hypomorphic alleles of hrp48, which encodes a member of the hnRNP A/B family and is implicated in control of P-element splicing. Significant reductions of mI and mII inclusion were also observed in heterozygotes for loss-of-function alleles of virilizer, fl(2)d, and crooked neck. The products of virilizer and fl(2)d are also required for Sxl autoregulation at the level of splicing; crooked neck encodes a protein with structural similarities to yeast-splicing factors Prp39p and Prp42p. Deletion of at least five other loci caused significant reductions in the inclusion of mI and/or mII (Burnette, 1999).

Coupled RT-PCR assays were used to analyze the pattern of Ubx alternative splicing in heterozygous third instar larvae and in adults. The isoform ratios in third instar larvae were in close agreement with those determined previously using nuclease protection assays. Types Ia and IIa are the predominant Ubx mRNAs and those lacking both mI and mII (isoforms IVa and b) make up only a small fraction of the total. Adults contain a significantly higher proportion of class IV mRNAs than larvae; this differs from previous reports and probably reflects the very early and narrow age distribution of the adults used in this study. It is important to note that the Ubx isoform ratios did not vary significantly between different wild-type strains nor between these and several control strains that carried different balancer chromosomes and irrelevant mutations. These results demonstrate that the mechanism that controls Ubx alternative splicing is robust, a conclusion that is consistent with the faithful conservation of Ubx isoform structure and expression among Drosophila species spanning 60 million years of evolution. The fact that the quantitative isoform pattern revealed by this assay is insensitive to considerable variation in genetic background highlights the significance of the effects described below for specific mutations and deficiencies. Although amplified Ubx cDNA fragments that contain mI but not mII (i.e., hypothetical isoforms IIIa and IIIb) should have the same length as isoforms IIa and IIb, such amplifiers would be expected to exhibit distinctly slower mobility due to the difference in nucleotide sequence (Burnette, 1999).

The products of Sxl, tra, and tra-2 are known regulators of alternative splicing decisions in Drosophila but they are not essential for processes other than sex determination (and dosage compensation, in the case of Sxl) because males that are null for these genes are viable and appear phenotypically normal. However, additional genes [fl(2)d, virilizer, and l(2)49Db] are required for correct control of alternative splicing decisions by Sxl are also essential for viability in both sexes; hence, their products may also have roles in other alternative splicing events. To determine whether these include the control of Ubx alternative splicing, it was asked whether the Ubx isoform ratios are altered in heterozygotes for mutations in these genes. In contrast to the stability described in the preceding section, the Ubx splicing pattern is altered significantly when the expression or function of virilizer or fl(2)d is reduced. The strongest effect is observed with virilizer, using a loss-of-function allele (vir³) that is recessive lethal in both sexes. In heterozygous larvae the proportion of Ubx class I mRNAs declines while that of classes II and IV increases. The proportion of class I that contains the B element is not altered. The increase in classes II and IV indicates that inclusion of both mI and mII is reduced but that the effect on mI exceeds that on mII. Inclusion of mI is also reduced in adults, although the effect was weaker than in larvae. More modest but statistically significant reductions of mI and mII inclusion are also observed in larvae heterozygous for the fl(2)d² mutation, which is also a loss-of-function allele that is recessive lethal in both sexes (Burnette, 1999).

hrp48 plays a critical role in the inclusion of mI and mII: hrp48 is a member of the hnRNP-A/B family of RNA-binding proteins and forms part of a protein complex that regulates splicing of intron 3 (IVS3) in P-element transcripts. Although repression of IVS3 splicing in somatic tissues is dictated by PSI, which is a soma-specific component of the regulatory complex, the hrp48 protein binds specifically to sequences within the cis-acting regulatory element in the RNA. hrp48 was originally identified as a general component of heterogeneous nuclear ribonucleoprotein particles and the hrp48 gene is essential for viability, so it must perform additional functions unrelated to P-element expression; these functions might include regulation of other splicing decisions. The five known mutant alleles of hrp48 are all P-element insertions in the upstream regulatory region and are not null. Nevertheless, inclusion of mI and mII in Ubx mRNAs is reduced markedly in larvae and adults heterozygous for the strong recessive lethal allele hrp48¹; weaker alleles, some of which are viable as homozygotes, have similar but more modest effects. The effect of hrp48 mutations resembles that of vir and fl(2) mutations: inclusion of mII is affected more weakly than mI, and the proportion of isoform I that contains the B element is not altered. Heterozygosity for hrp48¹ reduces inclusion of mI by 27%; this is the strongest effect observed for any mutation or deficiency in this study, indicating that normal levels of hrp48 are critical for inclusion of the internal exons, especially mI, in Ubx mRNAs (Burnette, 1999).

One enhancer, Df(1)64c18g, deletes the genes crooked neck (crn) and kurz (kz), which are located at 2F1 and are both candidate RNA-processing factors. The crn gene encodes a protein with 16 tetratrichopeptide repeats, a motif implicated in protein-protein interactions. Although CRN protein has been proposed to function as a transcription factor involved in cell cycle control, recent data show that it is closely related to the yeast splicing factors Prp39p and Prp42p, which associate with yeast U1 snRNP and are required for splicing. The kz gene encodes a protein with extensive homology to yeast ATP-dependent splicing factors Prp2p, Prp16p, and Prp22p. These proteins define a distinct subfamily of ATP-dependent putative RNA-helicases. Because mutant alleles of these genes are available, a direct test was carried out to see if deletion of one or both might be responsible for enhancement of the Ubx haltere phenotype and whether they affect the Ubx splicing pattern. Like the deficiency, two hypomorphic, recessive lethal alleles of crn (EA130 and RC63) act as dominant zygotic enhancers of Ubx¹⁹⁵/+ and Ubx^9.22/+. RT-PCR analysis shows that inclusion of mI, but not mII, is reduced significantly in larvae heterozygous for crn^EA130. The second allele, crn^RC63, has similar effects on the Ubx phenotype and splicing pattern. A recessive lethal allele of kz (DF942) behaves as a weak dominant enhancer of Ubx¹⁹⁵/+ and Ubx^9.22/+, but RT-PCR analyses does not reveal a significant dominant effect on the Ubx splicing pattern (Burnette, 1999).

The inclusion of mI and mII in Ubx mRNAs is regulated by competition between 5' splice sites that flank each of these exons after they are joined to E5'. As the RNA is transcribed, mI and subsequently mII are spliced constitutively to the upstream exon but can then be removed, together with the downstream intron, using an upstream 5' splice site within E5' or at the junction with this exon. For the majority of nascent RNAs (those initially spliced using 5' splice site a in E5'), a strong 5' splice site is regenerated at the junction between E5' and mI or mII that competes with the mI or mII 5' splice site located 51 nt downstream. For a minority of nascent RNAs (those initially spliced using 5' splice site b in E5') the a site is still present in E5' and can compete with the mI or mII 5' splice site located 78 nt downstream; use of the a site then removes the B element along with mI or mII. Developmental regulation of mI and mII inclusion is achieved by modulating the competition between the upstream and downstream 5' splice sites that flank these exons (Burnette, 1999).

Reduction of function in all of the factors identified in this work leads to reduced inclusion of mI (and in most cases also mII). This suggests roles in suppression of the upstream sites (which strongly match the 5' splice site consensus) or stimulation of the downstream sites (which match the consensus more weakly). It is interesting that three of the factors identified in this study that are required for inclusion of mI and mII in Ubx mRNAs may also be required for suppression of 5' splice site utilization in other RNAs: the functions of virilizer and fl(2)d are required for Sxl to repress splicing of the male-specific exon in its own RNA, and hrp48 is implicated as part of a complex that mediates repression of a 5' splice site in P-element RNA. In addition, heterozygosity for a null allele of sans-fille (snf^J210) does not alter the Ubx splicing pattern, but the antimorphic allele snf^e8H, which interferes with autoregulation of Sxl splicing, enhances the Ubx haltere phenotype and increases exclusion of mI and mII (Burnette, 1999 and references).

The products of virilizer, fl(2)d, and snf might function as parts of a complex that mediates active repression of 5' splice site utilization through interactions with U1 snRNP. Formation or stabilization of this repression complex could be directed to different target splice sites through the action of distinct factors that, like Sxl, bind to cis-acting regulatory signals and interact with components of the complex. An intriguing possibility is that hrp48 interacts (directly or indirectly) with a U1 snRnp/Snf/Vir/Fl(2)D complex to target suppression of splicing at the upstream sites that are used to remove mI. The strong reduction of mI inclusion (27%) observed in hrp48¹ heterozygotes suggests a critical role for hrp48 in modulating competition between the regenerated and downstream 5' splice sites that flank this exon. Although hrp48 is an hnRNP protein that probably binds nonspecifically to many RNAs, it is also known to form part of a specific complex that blocks use of the 5' splice site for the third intron of P-element RNA in somatic cells. This regulatory complex prevents U1 snRNP from binding at the 5' splice site and recruits it instead, nonproductively, to the more upstream of two overlapping pseudo-5' splice sites within the exon; hrp48 itself makes contact with the downstream pseudo-5' splice site, F2. Splicing of P-element IVS3 in a reporter transgene is partially derepressed in adult escapers homozygous for a semilethal hrp48 allele, indicating that hrp48 is necessary for efficient suppression of the 5' splice site. Hence, it may be significant that a sequence within mI that overlaps the regenerated 5' splice site matches F2 and flanking nucleotides at 8 of 10 positions; this sequence is conserved among four Drosophila species that diverged up to 60 million years but maintain identical regulation of mI inclusion. hrp48 might bind to this sequence and help to recruit U1 snRNP nonproductively to the regenerated 5' splice site at the E5'/mI junction; in intermediates where mI has been spliced to the b site of E5', this complex could also block access to the a site located 27 nt upstream. This would explain why the hrp48, vir, and fl(2)d mutations reduce mI inclusion but do not alter the proportion of class I mRNAs that contain the B element: failure to assemble the repression complex at the E5'/mI junction would allow inappropriate use of both the regenerated site (used to remove mI from E5'a/mI and E5'b/mI intermediates) and the a site (used to remove mI and the B element from E5'b/mI intermediates) (Burnette, 1999 and references).

The effect of hrp48, vir, and fl(2)d mutations on inclusion of exon mII, which does not contain an F2-like element, may not be the result of resplicing at the E5'/mII junction. The reduction of mII inclusion (detected as an increase in class IV mRNAs rather than a decrease in class II) could be explained if the repression complex must remain assembled at the E5'/mI junction to prevent subsequent removal of mI and mII together during splicing of intron 3. Intermediates from which mI is removed during splicing of intron 2 would retain mII. The net result would be an increase in both class II and class IV mRNAs, as observed. In addition, it is noted that the effect of hrp48 mutations on mI and mII inclusion is the opposite of what one would expect from the simple idea that hnRNP A/B proteins generally promote exon skipping (and use of upstream 5' splice sites), antagonizing a general effect of SR proteins that promote exon inclusion (or use of downstream 5' splice sites). The observations presented here are more consistent with a specific role for hrp48 acting through cis-regulatory elements to prevent resplicing of mI. It is more difficult to speculate on the roles of crn or the still-unidentified factors deleted by deficiencies that alter the Ubx splicing pattern. In principle, these could participate in repression of the regenerated 5' splice sites or stimulation of the competing downstream site. They could also be involved in interactions between mI and mII that seem to be required for effective use of the downstream 5' splice site located at the mI/intron 2 boundary. Although a weak homology to the homeodomain led to the proposal that the crooked neck protein functions as a transcription factor, its 16 tetratrichopeptide repeats form a distinct subfamily with those of Prp39p and Prp42p, two splicing factors from yeast that interact with U1 snRNP but appear not to bind RNA directly. A third yeast member of this group has been identified that has more extensive homology to crn ; it will be interesting to learn whether this also functions as a splicing factor (Burnette, 1999 and references).

Additional observations indicate that inclusion of mI is controlled by a complex regulatory switch employing multiple factors to balance positive and negative inputs acting on the upstream and perhaps downstream splice sites. A positive role for Rbp1 is suggested by studies of cis-acting elements within mI. Rbp1 is related to the mammalian SR proteins 9G8 and SRp20; it has been implicated in the control of dsx and fru RNA splicing as a component of exonic splicing enhancer complexes that assemble on cis-acting elements with the sex-specific factor Tra. Rbp1 is expressed widely in both sexes and is likely to play a role in the splicing of many RNAs. Mutations in mI at positions +11 and +14 downstream of the E5'/mI junction reduce the efficiency of the regenerated 5' splice site in vivo; these positions lie within a sequence that matches at 11 of 12 nucleotides a set of functionally important Rbp1-binding sites within the female-specific polypyrimidine tract of dsx RNA, suggesting that Rbp1 is required to stimulate use of the regenerated 5' splice site in Ubx. (Burnette, 1999 and references).

It is unlikely that the factors described here represent all of those with critical effects on Ubx splicing regulation. The analysis of deficiencies itself poses certain limitations: an effect on the Ubx haltere phenotype may be masked by the simultaneous deletion of a gene that encodes a negative regulator of Ubx expression or function or of two factors with opposite effect on the regulation of Ubx splicing. Furthermore, detailed molecular analyses by quantitative RT-PCR was performed only for those regions whose phenotypic interactions with Ubx were confirmed by overlapping deficiencies, but another 22 regions were tentatively identified by single deficiencies as containing strong haploinsufficient enhancers of Ubx and might harbor genes with important effects on splicing; thus the regions described above are probably only a subset of those that can be identified with this approach. Using the positional information provided by the deficiencies plus RT-PCR assays of the Ubx splicing pattern, it should be possible to identify specific mutations in the relevant gene(s) within any region of interest (Burnette, 1999).

The Drosophila microRNA iab-4 causes a dominant homeotic transformation of halteres to wings

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).

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).

A single Hox locus in Drosophila produces functional microRNAs from opposite DNA strands

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. 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. Several Hox transcripts overlap on opposite strands, providing evidence of extensive antisense transcription, including antisense transcripts for mir-iab-4 in flies and its mammalian equivalent mir-196. 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. 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: 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, 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. In contrast, mir-iab-4AS transcription was detected in the segments A8 and A9, where Abdominal-B (Abd-B) is known to be expressed. 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. 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 (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, 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. 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, 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, and iab-4 lies in one of the most extensively studied regions of the Drosophila genome. The frequent occurrence of antisense transcripts 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. 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, 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).

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