BIOLOGICAL OVERVIEW

The sex determination master switch, Sex-lethal, regulates the mitosis of early germ cells in Drosophila. Sex-lethal is an RNA binding protein that regulates splicing and translation of specific targets in the soma, but the germline targets are unknown. In an immunoprecipitation experiment aimed at identifying targets of Sex-lethal in early germ cells, the RNA encoded by gutfeeling, the Drosophila homolog of ornithine decarboxylase (ODC) antizyme (Salzberg, 1996), was isolated (Vied, 2003).

Mammalian Antizyme negatively regulates ODC catalytically as well by directing the inactivated enzyme to the proteasome for degradation. This negative regulation of ODC is part of a feedback loop that controls the levels of polyamines within the cell. Translation of Antizyme is dependent on ribosomal frameshifting, which is promoted by high levels of polyamines. As polyamine levels in the cell rise, more Antizyme is synthesized, leading to the turnover of ODC. Polyamines have been implicated in many processes, including cell growth, transcription, and differentiation (Vied, 2003 and references therein). In mammals Antizyme and ubiquitin are thought to be respectively two types of proteasome targeting devices that mark proteins for both ubiquitin-independent and ubiquitin-dependent degradation by the 26 S proteasome (see Gruendler, 2001).

Drosophila gutfeeling interacts genetically with Sex-lethal. It is not only a target of Sex-lethal, but also appears to regulate the nuclear entry and overall levels of Sex-lethal in early germ cells. This regulation of Sex-lethal by gutfeeling appears to occur downstream of the Hedgehog signal. Gutfeeling appears to regulate the nuclear entry of Cyclin B as well. Hedgehog, Gutfeeling, and Sex-lethal function to regulate Cyclin B, providing a link between Sex-lethal and mitosis (Vied, 2003).

Sxl is necessary for female germ cell development regulating mitosis and the female-specific function of recombination. The targets and mechanism by which Sxl controls these processes are not known. Mitotic germ cells reside in the germarium, which is the anteriormost structure of the ovariole. Each of the 14-16 ovarioles of the ovary contains 2-3 stem cells, which divide to produce a cystoblast and another stem cell. Cystoblasts undergo 4 synchronous mitotic divisions with incomplete cytokinesis to form an interconnected 16-cell cyst. One of these 16 cystocytes becomes the oocyte, and the remaining 15 become nurse cells. This occurs as the 16-cell cyst progresses down the germarium. Toward the posterior of the germarium cells of somatic origin, the follicle cells, surround each cyst. This encapsulation is followed by the cyst pinching off from the germarium to produce an egg chamber. The egg chamber moves down the ovariole as it matures into an egg (Vied, 2003 and references therein).

Given the requirement of Sxl in the germline, attempts were made to identify its RNA targets by immunoprecipitation of the protein. To enrich for early germ cells, where Sxl impacts mitosis, advantage was taken of specific female sterile alleles (the Sxl^fs alleles) which arrest ovarian development at this stage. In these cells, Sxl protein is expressed at high levels and localized primarily in the cytoplasm as seen in the stem cells and cystoblasts. The mutant Sxl protein fails to undergo the dramatic downregulation that normally occurs as the germ cells mature. One of the RNA molecules identified in the immunoprecipitations is encoded by the gutfeeling (guf) gene, the Drosophila ornithine decarboxylase (ODC) antizyme (Vied, 2003).

The connection of Guf with Sxl provides a potential link between Sxl and mitosis. It has been demonstrated in vertebrate cells that both ornithine decarboxylase and polyamine levels undergo changes that coincide with cell cycle transitions and that depletion of polyamines results in a cell cycle arrest. The polyamine synthetic pathway has also been shown to regulate Cyclin B mRNA levels in vertebrate cells. When ODC is inhibited, cyclin B mRNA levels increase. More directly, Guf affects the nuclear localization of Cyclin B, as it does of Sxl. In addition, the data show that the nuclear entry of Cyclin B depends on Sxl. Typically, Cyclin B regulates cell cycle transitions, but this does not appear to be the case in germ cells. Unlike Cyclin A, whose levels change with the cell cycle, Cyclin B levels remain constant in early germ cells. Additionally, Cyclin A associates with the fusome during the G2 phase of the cell cycle, while Cyclin B does not. This suggests that Cyclin A and Cyclin B regulate early germ cell mitosis by different mechanisms. Overexpression of Cyclin B (or Cyclin A) results in only one extra round of cystocyte divisions to produce a 32-cell cyst, and it has been proposed that Cyclin B (and Cyclin A) regulate specialized cystocyte divisions. Cyclin B is downregulated at approximately the 2-cell cyst stage (the same as Sxl) and is not detected again until the 16-cell stage. This timing may reflect a specific requirement for Cyclin B in one of the 4 mitoses that generate the 16-cell cyst. The dependence of Cyclin B nuclear entry on Sxl suggests how Sxl might regulate germ cell mitosis (Vied, 2003).

The genetic data suggest that the effect of Guf on Sxl and Cyclin B is downstream of the Hh signal. In early germ cells, Sxl is in a complex with the cytoplasmic components of the Hh signaling pathway. While the data do not provide evidence to the point of action, it is suggestive that Guf promotes the disassembly of this complex with Sxl. Given that Antizyme (Guf) binds directly to the vertebrate ODC and Smad1 proteins and targets them to the proteasome (reviewed by Coffino, 2001a and b and Hoyt, 2003), it is tempting to speculate that the disassembly of the Hh cytoplasmic components involves targeting of one or more of the components to the proteasome. Additionally, it has been demonstrated that the inhibitory cytoplasmic NF-kappaB complex can be dissociated by changes in polyamine levels. Inhibiting ODC and thus depleting polyamine levels, a condition equivalent to Antizyme overexpression, leads to the proteolysis of Ikappa-Balpha. NF-kappaB dissociates from the cytoplasmic complex and enters the nucleus where it affects transcription. These effects are analogous to the role proposed for Guf in dissociating the components of the Hh cytoplasmic complex (Vied, 2003).

In its effects on Sxl, guf appears to act downstream of Hh and upstream of the cytoplasmic components. If guf functions downstream of Hh, it does not appear to do so in all Hh-regulated ovarian processes. This is exemplified by the lack of an effect on the turnover of the fusome. Also, guf does not affect the known role of Hh in regulating follicle cell proliferation. Finally, a connection between Cyclin B and the Hh signaling pathway has been previously described, but in a different context. Patched 1 (Ptc 1), which is one of two Ptc proteins in mammals, has been shown to directly interact with Cyclin B, inhibiting its nuclear translocation until cells were exposed to the Hh signal. Whether Ptc directly interacts with Cyclin B in Drosophila remains to be determined. These results, nevertheless, provide an intriguing parallel linking the Hh signal and Guf to the nuclear translocation of Sxl and Cyclin B (Vied, 2003).

GENE STRUCTURE

The Drosophila guf locus appears to have four promoters because four different cDNAs are generated. Each cDNA has a different 5' exon with the translation start site (except for the second 5' exon, which does not contain a start codon) which is spliced to two common exons, the first of which contains the translation frameshifting site (Ivanov, 1998). Interestingly, the polypyrimidine tract at the 3' splice site of the first common exon has a run of eight Uridines (U). This is a feature found in all established Sxl targets: msl-2, tra, and Sxl. Additionally, guf has several long polyU tracts in the introns of all its transcripts. For splicing regulation in vivo, Sxl shows a requirement of at least seven Us at its binding site, while runs of six Us gives compromised regulation. In vitro, shortening of target polyU tracts below six severely decreases the affinity of Sxl protein for its RNA (Vied, 2003).

In the 3' UTR short, six nucleotide-long, polyU runs exist in the mature guf message. This raised the question of whether Sxl regulates guf primarily through splicing or translation control. To address this, Sxl was immunoprecipitated from extracts of both wild-type and Sxl^f4 ovaries, and the RNA in the immunoprecipitates was examined by RT-PCR with guf-specific primers. The primers span the two introns of the smallest guf transcript, so the size of the PCR products allows for distinguishing between spliced and unspliced forms of guf RNA (Vied, 2003).

A Drosophila antizyme with high homology to the sequence for mammalian antizyme (ornithine decarboxylase antizyme) has been reported. Homology of this coding sequence to its mammalian antizyme counterpart also extends to a 5' open reading frame (ORF) which encodes the amino-terminal part of antizyme and overlaps the +1 frame (ORF2) that encodes the carboxy-terminal three-quarters of the protein. Ribosomes shift frame from the 5' ORF to ORF2 with an efficiency regulated by polyamines. At least in mammals, this is part of an autoregulatory circuit. The shift site and 23 of 25 of the flanking nucleotides (which are likely important for efficient frameshifting) are identical to their mammalian homologs. In the reverse orientation, within one of the introns of the Drosophila antizyme gene, the gene for snRNP Sm D3 is located. Previously, it was shown that two closely linked P-element transposon insertions caused the gutfeeling phenotype of embryonic lethality and aberrant neuronal and muscle cell differentiation. The present work shows that defects in either snRNP Sm D3 or antizyme, or both, are likely causes of the phenotype (Ivanov, 1998).

Programmed frameshifting is known to occur in single-celled eukaryotes (yeast and protozoa) and in Xenopus but has not previously been discovered in any intermediate organism. The finding of antizyme programmed frameshifting in Drosophila provides the opportunity to study the evolution of this recoding event, which involves a transitory alteration of the rules of readout of the genetic code. The pseudoknot, a 3' mRNA message feature that is an important stimulator for the recoding signal in mammalian systems, is not recognizable in Drosophila, and the identification of its presumed alternative is of major interest. Of the three known RNA elements that stimulate antizyme frameshifting in mammalian cells, the other two are present in guf. One of these is the UGA stop codon of ORF1. It is interesting that even though in vitro translation experiments have shown that the other two stop codons (UAG and UAA) are almost as effective in stimulating frameshifting in decoding mammalian antizyme sequences so far all eukaryotic antizyme genes have UGA as the stop codon of ORF1. Another stimulatory element, a sequence immediately 5' of the UGA stop codon, is also likely to be present in the D. melanogaster antizyme sequence. Of 18 nucleotides 5' of the UGA stop codon, 16 are identical for guf and the rat antizyme sequence (with one of the two mismatches being an antizyme polymorphism). This level of 5' sequence homology is striking and provides a clear indication that this region plays an important role in antizyme frameshifting. The apparent absence of an RNA pseudoknot 3' of the UGA stop codon of guf1 is puzzling. The nucleotides within the stems of this pseudoknot are absolutely conserved among all known vertebrate antizyme sequences. Despite the apparent absence of a 3' pseudoknot, there is nucleotide sequence homology between this region of guf and vertebrate antizyme sequences. Perhaps some other RNA structure is present in this region of guf. Indirect evidence supports such a hypothesis. Comparison of D. melanogaster and D. virilis antizyme nucleotide sequences reveals that the 53 nt between the UGA of the putative frameshift site and intron 2 (a region most likely to contain a stimulatory 3' RNA structure) are completely conserved between the two species, even though the conservation of the rest of the known exonic sequences is only 75%. Computer programs predict several stem loops in this region, but the relevance of any of them to translational frameshifting is unknown. Even though additional frameshift-stimulatory elements in the Drosophila antizyme sequence are not obvious, there is a strong indication that there is more to the guf frameshift site than the 28-nt region homologous to the mammalian antizyme frameshift site. The 28 nt alone cannot stimulate more than 3% frameshifting in vitro. The data for the in vitro translation of guf strongly suggests that the frameshift efficiency in that system is much higher than 3%, thus implying the existence of additional frameshift-stimulatory signals in Drosophila antizyme mRNA (Ivanov, 1998).

cDNA clone length - 2163

Bases in 5' UTR - 1260

Exons - 3

Bases in 3' UTR - 242

PROTEIN STRUCTURE

Amino Acids - 254

Structural Domains

The guf cDNA contains a single long open reading frame that is preceded by an uncommonly long untranslated leader (1260 bp). The predicted 20.8 X l0³ Mr Guf protein is hydrophilic and acidic (predicted PI = 4.39) containing no hydrophobic regions that may correspond to a transmembrane domain or a signal sequence. Search of the PROSITE dictionary of protein sites and patterns did not identify any potential sites for posttranslational modifications such as phosphorylation. BLAST searches revealed a significant sequence similarity between Guf and the vertebrate ODC antizyme (OAZ). The sequence similarity between Guf and OAZ spans almost the entire length of the Guf protein excluding the first 30 amino acids. The overall homology is 24.4% identity and 42% similarity. The highest degree of conservation, 43% identity and 61% similarity, is found in the region of amino acids 100-157. Interestingly, a 22-amino acid stretch that was shown by Ichiba (1994) to be essential for the binding of OAZ to ODC is found in the core of this domain (amino acids 116-137). In contrast, the short stretch that was found by Ichiba to be most important for the ODG destabilizing activity of OAZ (amino acids 74-79) is not conserved (Salzberg, 1996).

date revised: 12 May 2003

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