Evolutionary Conservation of the Structure and Expression of Alternatively Spliced Ultrabithorax Isoforms from Drosophila

In Drosophila melanogaster, alternatively spliced mRNAs from the homeotic gene Ultrabithorax (Ubx) encode a family of structurally distinct homeoprotein isoforms. The developmentally regulated expression patterns of these isoforms suggest that they have specialized stage- and tissue-specific functions. To evaluate the functional importance of UBX isoform diversity and gain clues to the mechanism that regulates processing of Ubx RNAs, we have investigated whether the Ubx RNAs of other insects undergo similar alternative splicing. We have isolated and characterized Ubx cDNA fragments from D. melanogaster, Drosophila pseudoobscura, Drosophila hydei and Drosophila virilis, species separated by as much as 60 million years of evolution, and have found that three aspects of Ubx RNA processing have been conserved. (1) These four species exhibit identical patterns of optional exon use in a region adjacent to the homeodomain. (2) These four species produce the same family of UBX protein isoforms with identical amino acid sequences in the optional exons, even though the common amino-proximal region has undergone substantial divergence. The nucleotide sequences of the optional exons, including third positions of rare codons, have also been conserved strongly, suggesting functional constraints that are not limited to coding potential. (3) The tissue- and stage-specific patterns of expression of different UBX isoforms are identical among these Drosophila species, indicating that the developmental regulation of the alternative splicing events has also been conserved. These findings argue for an important role of alternative splicing in Ubx function. We discuss the implications of these results for models of UBX protein function and the mechanism of alternative splicing.     

Transvection in the Drosophila Ultrabithorax Gene: A Cbx(1) Mutant Allele Induces Ectopic Expression of a Normal Allele in Trans

In wild-type Drosophila melanogaster larvae, the Ultrabithorax (Ubx) gene is expressed in the haltere imaginal discs but not in the majority of cells of the wing imaginal discs. Ectopic expression of the Ubx gene in wing discs can be elicited by the presence of Contrabithorax (Cbx) gain-of-function alleles of the Ubx gene or by loss-of-function mutations in Polycomb (Pc) or in other trans-regulatory genes which behave as repressors of Ubx gene activity. Several Ubx loss-of-function alleles cause the absence of detectable Ubx proteins (UBX) or the presence of truncated UBX lacking the homeodomain. We have compared adult wing phenotypes with larval wing disc UBX patterns in genotypes involving double mutant chromosomes carrying in cis one of those Ubx mutations and the Cbx1 mutation. We show that such double mutant genes are (1) active in the same cells in which the single mutant Cbx1 is expressed, although they are unable to yield functional proteins, and (2) able to induce ectopic expression of a normal homologous Ubx allele in a part of the cells in which the single mutant Cbx1 is active. That induction is conditional upon pairing of the homologous chromosomes (the phenomenon known as transvection), and it is not mediated by UBX. Depletion of Pc gene products by Pc3 mutation strongly enhances the induction phenomenon, as shown by (1) the increase of the number of wing disc cells in which induction of the homologous allele is detectable, and (2) the induction of not only a paired normal allele but also an unpaired one.     

Trans-acting factors required for inclusion of regulated exons in the Ultrabithorax mRNAs of Drosophila melanogaster

Alternatively spliced Ultrabithorax mRNAs differ by the presence of internal exons mI and mII. Two approaches were used to identify trans-acting factors required for inclusion of these cassette exons. First, mutations in a set of genes implicated in the control of other alternative splicing decisions were tested for dominant effects on the Ubx alternative splicing pattern. To identify additional genes involved in regulation of Ubx splicing, a large collection of deficiencies was tested first for dominant enhancement of the haploinsufficient Ubx haltere phenotype and second for effects on the splicing pattern. Inclusion of the cassette exons in Ubx mRNAs was 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. Possible roles of identified factors are discussed in the context of the resplicing strategy for generation of alternative Ubx mRNAs.     

Integration of RNA processing and expression level control modulates the function of the Drosophila Hox gene Ultrabithorax during adult development

Although most metazoan genes undergo alternative splicing, the functional relevance of the majority of alternative splicing products is still unknown. Here we explore this problem in the Drosophila Hox gene Ultrabithorax (Ubx). Ubx produces a family of six protein isoforms through alternative splicing. To investigate the functional specificity of the Ubx isoforms, we studied their role during the formation of the Drosophila halteres, small dorsal appendages that are essential for normal flight. Our work shows that isoform Ia, which is encoded by all Ubx exons, is more efficient than isoform IVa, which lacks the amino acids coded by two small exons, in controlling haltere development and regulating Ubx downstream targets. However, our experiments also demonstrate that the functional differences among the Ubx isoforms can be compensated for by increasing the expression levels of the less efficient form. The analysis of the DNA-binding profiles of Ubx isoforms to a natural Ubx target, spalt, shows no major differences in isoform DNA-binding activities, suggesting that alternative splicing might primarily affect the regulatory capacity of the isoforms rather than their DNA-binding patterns. Our results suggest that to obtain distinct functional outputs during normal development genes must integrate the generation of qualitative differences by alternative splicing to quantitative processes affecting isoform protein expression levels.     

The localization and regulation of Antennapedia protein expression in Drosophila embryos

The homeotic Antennapedia (Antp) gene of Drosophila is required for the normal differentiation of the thoracic segments during embryonic development and metamorphosis. Antibodies to a recombinant Antp protein were used to localize the protein in whole mount embryos. Antp is expressed in the nuclei of cells of the thoracic embryonic epidermis and several segments of the ventral and peripheral nervous systems. Analysis of Antp expression in mutant embryos revealed three levels of Antp regulation by genes of the bithorax complex, pleiotropic homeotic loci, and Antp itself. The distributions of the Antp and the Ultrabithorax (Ubx) proteins in doubly-labeled embryos suggest that the Ubx protein may be one direct negative regulator of Antp gene expression.     

Functional dissection of Ultrabithorax proteins in D. melanogaster

Expression of Ultrabithorax (UBX) proteins via a heat-inducible promoter generated homeotic transformations of segmental identities in the embryonic cuticle and peripheral nervous system (PNS) of Drosophila and transformed antennae into legs in the adult. The embryonic transformations were used to determine the identity functions of members of the UBX family and UBX mutant forms. Whereas UBX forms I and IV each induced the cuticle transformations, only form I induced the PNS transformations. Analysis of the transformations generated by UBX deletions and by a chimeric Ultrabithorax-Antennapedia protein demonstrated that the majority of the UBX identity information is contained within the C-terminal, homeodomain-containing portion of the protein. Implications of these results for how homeotic proteins select particular metameric identities are discussed.     

Protein products of the bithorax complex in Drosophila

A sequence from the Ubx 5' exon in the bithorax complex of Drosophila melanogaster was expressed as a fusion protein in bacteria. This protein was used to raise rabbit antisera and monoclonal antibodies. These antibodies detect antigens that, on protein blots and by immunofluorescence on whole mounts of imaginal discs, show the predicted segmental distribution of Ubx products. These products are predominantly, if not totally, localized in the cell nucleus. In the embryonic nervous system nuclei are labeled from the second thoracic segment to the eighth abdominal segment. There is no labeling in homozygous Df bxd100 embryos.     

UBX domain-containing proteins are involved in lipid homeostasis and stress responses in Pichia pastoris

Ubiquitin regulatory X (UBX) domain-containing proteins constitute a family of proteins and are substrate adaptors of AAA ATPase Cdc48. UBX proteins can bind to the N-terminal region of Cdc48 to perform endoplasmic reticulum associated protein degradation (ERAD). In this study, we identified two UBX domain-containing proteins, Ubx1 and Ubx2, in Pichia pastoris and found that the two proteins could recover the growth defect of Saccharomyces cerevisiae in ubx2Δ. Our results revealed that Ubx1 and Ubx2 play critical roles in synthesis of unsaturated fatty acids by affecting Spt23. In addition, the results demonstrated that both Ubx1 and Ubx2 are involved in lipid droplet formation and protein degradation. Deletion of UBX1 led to increased sensitivity to oxidative stress and disruption of UBX2 impaired cell viability under osmotic stress. The phenotypes of ubx1Δ+UBX2, ubx2Δ+UBX1 and ubx1Δubx2Δ and RNA-seq data suggested that Ubx1 and Ubx2 play different roles in cell functions, and the roles of Ubx1 may be more numerous than Ubx2. In summary, our findings provide new insights into the relationship between lipid homeostasis and cell functions in the oil-producing organism P. pastoris.     

Ubx2 links the Cdc48 complex to ER-associated protein degradation

Endoplasmic reticulum (ER)-associated protein degradation requires the dislocation of selected substrates from the ER to the cytosol for proteolysis via the ubiquitin-proteasome system. The AAA ATPase Cdc48 (known as p97 or VCP in mammals) has a crucial, but poorly understood role in this transport step. Here, we show that Ubx2 (Sel1) mediates interaction of the Cdc48 complex with the ER membrane-bound ubiquitin ligases Hrd1 (Der3) and Doa10. The membrane protein Ubx2 contains a UBX domain that interacts with Cdc48 and an additional UBA domain. Absence of Ubx2 abrogates breakdown of ER proteins but also that of a cytosolic protein, which is ubiquitinated by Doa10. Intriguingly, our results suggest that recruitment of Cdc48 by Ubx2 is essential for turnover of both ER and non-ER substrates, whereas the UBA domain of Ubx2 is specifically required for ER proteins only. Thus, a complex comprising the AAA ATPase, a ubiquitin ligase and the recruitment factor Ubx2 has a central role in ER-associated proteolysis.     

Identification of chicken nebulin isoforms of the 31-residue motifs and non-muscle nebulin

Nebulin is a very large (M(r) 600-900kDa) actin-binding protein that is specific to skeletal muscle, and which is thought to act as a molecular template that regulates the length of sarcomere thin filaments. The 31-residue motif of nebulin contains a unique PEhXRVKXNQ consensus sequence. We have previously identified 11 different human nebulin isoforms of these 31-residue motifs. Here we present the identification of seven different isoforms (types II, III, IVa, IVb, V, VI, and X) of the 31-residue motifs in 15-day-old chicken embryo breast muscle. Isoform types II and III are also expressed in the brain, and type III is also detected in the heart, stomach, and liver. Chicken nebulin contains 11 copies of the 31-residue motif (R1a/b, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11), whereas human nebulin contains 13 copies. We confirmed the expression of nebulin in the heart, stomach, and brain in 15-day-old chicken embryos by immunofluorescence microscopy. The presence of nebulin in brain was further confirmed by in situ hybridization. These data suggest that there is even more diversity in nebulin isoforms than was previously known; this diversity likely contributes to the distinct actin filament architecture of different tissues.     

hnRNP A1 and secondary structure coordinate alternative splicing of Mag

Myelin-associated glycoprotein (MAG) is a major component of myelin in the vertebrate central nervous system. MAG is present in the periaxonal region of the myelin structure, where it interacts with neuronal proteins to inhibit axon outgrowth and protect neurons from degeneration. Two alternatively spliced isoforms of Mag mRNA have been identified. The mRNA encoding the shorter isoform, known as S-MAG, contains a termination codon in exon 12, while the mRNA encoding the longer isoform, known as L-MAG, skips exon 12 and produces a protein with a longer C-terminal region. L-MAG is required in the central nervous system. How inclusion of Mag exon 12 is regulated is not clear. In a previous study, we showed that heteronuclear ribonucleoprotein A1 (hnRNP A1) contributes to Mag exon 12 skipping. Here, we show that hnRNP A1 interacts with an element that overlaps the 5′ splice site of Mag exon 12. The element has a reduced ability to interact with the U1 snRNP compared with a mutant that improves the splice site consensus. An evolutionarily conserved secondary structure is present surrounding the element. The structure modulates interaction with both hnRNP A1 and U1. Analysis of splice isoforms produced from a series of reporter constructs demonstrates that the hnRNP A1-binding site and the secondary structure both contribute to exclusion of Mag exon 12.     

The Ubx2 and Ubx3 cofactors direct Cdc48 activity to proteolytic and nonproteolytic ubiquitin-dependent processes

Valosin-containing protein, VCP/p97 or Cdc48, is a eukaryotic ATPase involved in membrane fusion, protein transport, and protein degradation. We describe two proteins, Ubx2 and Ubx3, which interact with Cdc48 in fission yeast. Ubx3 is the ortholog of p47/Shp1, a previously described Cdc48 cofactor involved in membrane fusion, whereas Ubx2 is a novel protein. Cdc48 binds the UBX domains present in both Ubx2 and Ubx3, indicating that this domain is a general Cdc48-interacting module. Ubx2 and Ubx3 also interact with ubiquitin chains. Disruption of the ubx3(+)-gene causes both temperature and canavanine sensitivity and stabilizes some ubiquitin-protein conjugates including the CDK inhibitor Rum1, but not a model substrate of the ER-degradation pathway. Moreover the ubx3 null displays synthetic lethality with a pus1 null mutant, a multiubiquitin binding subunit of the 26S proteasome. In contrast, the ubx2 null mutant did not display any obvious protein-degradation phenotype. In conclusion Ubx3/p47 is not, as previously thought, only important for membrane fusion; it's also important for the specific degradation of a subset of cell proteins. Our genetic analyses revealed that Ubx3/p47 functionally parallels a substrate receptor of the 26S proteasome, Pus1/Rpn10, indicating that the Cdc48-Ubx3 complex is involved in delivering substrates to the 26S proteasome.     

Trypsin Inhibitor Polymorphism: Multigene Family Expression and Posttranslational Modification

Trypsin inhibitors from winter pea seeds (c.v. Frilene) have been purified and shown to consist of six protease inhibitors (PSTI I, II, III, IVa, IVb, and V). Based on amino acid composition, molecular mass, and N-terminal sequence, the six inhibitors are closely related to one another and belong to the Bowman–Birk family of inhibitors. To define the relations among them, molecular mass and amino acid composition of peptides obtained from digestion with trypsin were determined. The sequence and the biosynthetic mechanism of the isoform formation have been partially resolved for four major isoforms. Two isoinhibitor forms (PSTI IVa, IVb) in pea seeds are due to expression of two distinct genes; PSTI IVa has four amino acid replacements when its sequence is compared with the sequence of PSTI IVb. Two others (PSTI I, II) result from posttranslational proteolytic cleavage of nine C-terminal residues of forms PSTI IVa and IVb, respectively.     

Tissue- and stage-specific control of homeotic and segmentation gene expression in Drosophila embryos by the polyhomeotic gene

The distributions of the products of the homeotic genes Sex combs reduced (Scr) and Ultrabithorax (Ubx) and of the segmentation genes, fushi tarazu (ftz), even skipped (eve) and engrailed (en) have been monitored in polyhomeotic (ph) mutant embryos. None of the genes monitored show abnormal expression at the blastoderm stage in the absence of zygotic ph expression. Both Scr and Ubx are ectopically expressed in the epidermis of ph embryos, confirming the earlier proposal, based on genetic analysis, that ph+ acts as a negative regulator of Antennapedia (ANT-C) and bithorax (BX-C) complex genes. At the shortened germ band stage, en is also ectopically expressed, mainly in the anterior region of each segment. In contrast to these effects in the epidermis, the expression of en, Ubx, Scr and ftz is largely or completely suppressed in the central nervous system, whereas eve becomes ectopically expressed in most neurones.