Gene name - virilizer
Cytological map position - 59D8--11
Function - splice factor
Symbol - vir
FlyBase ID: FBgn0003977
Genetic map position - 2-103.3
Classification - transmembrane protein with conserved domain
Cellular location - nuclear membrane
virilizer (vir) influences sex determination in Drosophila. Vir is a putative splicing factor involved in the post-transcriptional regulation of Sex lethal (Sxl). The regulation of Sxl transcription and translation is complex and occurs in two steps. First, activation of the gene is transcriptional and relies on an establishment promoter (Pe), which is transcribed only around blastoderm stage in response to a female X:A ratio (XX:AA). Transcripts derived from Pe give rise to early Sxl protein. Transcription from this promoter ends shortly after blastoderm stage when a constitutive promoter (Pm) becomes active in both sexes. From then on, Sxl expression is post-transcriptionally regulated. In the presence of early Sxl, pre-mRNA derived from Pm is female-specifically spliced by skipping exon 3. This exon contains STOP codons in all three reading frames, and therefore functional late Sxl cannot be produced if the exon is present in the mRNA. Late Sxl is used continuously to splice Sxl pre-mRNA in the female mode, and an autoregulatory feedback loop is established. Sxl directs exon skipping by cooperatively binding to multiple uridine-rich stretches within and around the regulated exon. A male primary signal (X:AA) does not activate Pe, and early Sxl is not produced. As a consequence, exon 3 is retained in the Sxl mRNAs when Pm becomes active in males (Niessen, 2001 and references therein).
In addition to Sxl itself and vir, two other genes are needed for Sxl autoregulation: sans fille (snf) and female-lethal-2-d [fl(2)d]. Female-specific mutations exist in vir, snf and fl(2)d that affect the expression of Sxl, but most alleles are lethal to both sexes, suggesting also a more general vital role. snf encodes a nuclear protein with functional and sequence similarity to the mammalian U1A and U2B" snRNP proteins. Sxl physically interacts with SNF via its RRM 1 (RNA recognition motif 1). Therefore, snf is probably directly involved in splice site recognition. fl(2)d encodes a novel nuclear protein with an amino terminal HisGlu-rich domain that is often found in transcriptional regulators. FL(2)D is expressed at all stages of development in both sexes. For virilizer (the subject of this overview) three phenotypic classes of alleles are known. One temperature-sensitive allele, vir1ts, transforms XX animals into intersexes at the restrictive temperature of 29°; this mutation shows that vir is involved in sex determination. A different allele, vir2f, is an XX-specific lethal that interferes with dosage compensation and sex determination. Alleles in the third and largest class kill both sexes in the third larval instar, implying a vital function of vir unrelated to sex. In cell clones, the lethal alleles cause sexual transformation of XX cells into male structures, and in trans with vir2f, they cause female-specific lethality, suggesting that they lack the function required for female-specific expression of Sxl. Indeed, in XX animals mutant for vir2f, Sxl pre-mRNA is spliced in the male mode, indicating that vir is required for the female-specific splicing of Sxl transcripts (Hilfiker, 1995 and Niessen, 2001 and references therein).
In contrast to the complex effects of vir mutations, the gene itself produces only a single transcript that encodes a large protein. The structure of the putative Vir protein has prompted speculation about the molecular mechanism by which vir affects the expression of Sxl and other vital genes. The prediction of a transmembrane domain, the nuclear localization of Vir, and the domain structure suggest that Vir may be a member of a new class of splice regulators (Niessen, 2001).
Sxl is not the only gene that requires the function of vir. Female-specific expression of transformer and msl-2 depend on vir even when Sxl protein is present. This is seen in XX animals mutant for vir2f, which can be partially rescued by the constitutive allele SxlM4. However, such females are strongly masculinized because the transcripts of tra are not efficiently spliced in the female mode, and their low survival rate points to only partial repression of msl-2 (Hilfiker, 1995). That vir is directly involved in splicing of Sxl and tra pre-mRNA cannot be ruled out, but Vir is not an essential component of the general splicing machinery because even amorphic vir alleles allow growth and differentiation of imaginal discs. Instead, Vir might be used to splice a subset of pre-mRNAs. Among these are the transcripts of Sxl and tra, which are both needed only in females. Another target is the primary transcript of the Ubx gene (Burnette, 1999). In the absence of Vir or FL(2)D, microexons mI and mII are not efficiently included in Ubx transcripts. There are probably other essential genes whose splicing depends on vir and fl(2)d. The sex-unspecific lethality associated with amorphic alleles of vir could be caused by the failure to splice the pre-mRNAs of such targets in the correct manner (Niessen, 2001).
How does vir perform the three functions associated with it, namely, a role in sex determination, in dosage compensation, and in a vital process unrelated to sex? The effects of vir mutation on sex determination and dosage compensation are easily explained by the requirement of vir for the female-specific splicing of transcripts of Sxl, which controls both these processes (Hilfiker, 1995). XX animals mutant for the female-specific allele vir2f express the gene Sxl in the male mode. Such animals could lack one protein variant that is necessary for female-specific splicing of Sxl pre-mRNA. vir alleles lethal for XX and XY animals might in addition miss another protein variant required for the vital function. Both variants would be missing in the latter class of mutants, which are deficient for the function required for Sxl autoregulation as well as for the vital function. Molecular analysis, however, shows that vir, despite its complex function, produces only a single transcript that probably encodes a single protein. The two XX-specific vir alleles, vir2f (1283 M-K) and vir1ts (1423 E-K), cause amino acid substitutions that lie relatively close to one another. These mutations may define a domain in Vir that is essential for female-specific splicing of Sxl pre-mRNAs, but not for the vital function of the protein. This hypothesis implies that a second domain exists that harbors the vital function. However, the genetic data are more in favor of a single protein with a single molecular function. Except for the two female-specific alleles, all the 34 vir mutations isolated so far affect the regulation of Sxl and the vital function. The three lethal alleles (vir4, vir22, and vir23) all cause large carboxy-terminal truncations of Vir. These truncations also remove the region mutated in vir1ts and in vir2f: this is consistent with the lethal alleles also being deficient in Sxl autoregulation (Hilfiker, 1995 and Niessen, 2001).
Different threshold requirements might exist for vir in Sxl autoregulation and in the vital process, the former being more sensitive than the latter. The female-specific and the lethal mutations may represent weaker (hypomorphic) and stronger (amorphic) alleles of the gene (Niessen, 2001).
Loss-of-function mutations in vir that cause male-specific splicing of the Sxl pre-mRNA in XX animals suggest a nuclear function for vir in the regulation of Sxl (Hilfiker, 1995). Consistent with this idea, Vir contains an NLS, and a Vir fusion protein locates to the nucleus in Drosophila tissue culture cells. This result, however, does not yet reveal the specific molecular role of the protein. The amino acid sequence of Vir contains a putative transmembrane domain at the amino terminus. Three regions in the protein have a high probability of forming a coiled coil, which, in other proteins, was demonstrated to serve as an interface for homo- or hetero-dimeric binding. Furthermore, a region is found in Vir with similarity to domain 6303 of the ProDom database. This domain is found in several RNA and DNA helicases, in translation initiation factor 2 (IF2) from Borrelia, and in ribonucleoproteins. The member of the family with the highest similarity to Vir within this domain is a human RNA-binding protein (TrEMBL: Q15415) that is thought to be involved in RNA processing or translation. However, there are also striking similarities between Vir and ROG_Human, which is the human hnRNP G. Domain 6303 is present in 4 other hnRNPs as well as hnRNP G: hnRNP A1, D1, R, and U all contain this domain. The proposed functions of these hnRNPs are diverse. hnRNP A1 is involved in RNA splicing, mRNA transport, and telomere biogenesis; hnRNP D1 is thought to be involved in transcription, while hnRNP U has a function in nuclear retention. No functions have yet been assigned to hnRNP G and R (Niessen, 2001 and references therein).
To test if this domain is preferentially present in proteins that interact with RNA, a pattern was derived from domain 6303 and it was used to scan various data banks (using the PatternFind Server at ISREC). Thirty-three proteins were recovered that contained the pattern. Fifteen of them are indeed predicted to interact with RNA (e.g., rRNA methyltransferases, RNA helicases, snRNPs, poly(A)binding proteins) and three are predicted to bind to DNA. Because domain 6303 is also found in proteins without known nucleic acid binding ability and is not present in some proteins that do bind to nucleic acids, it is unlikely that this domain confers binding itself. Rather, it might act as an interface for protein interactions (Niessen, 2001).
The prediction of a transmembrane domain, the nuclear localization of Vir, and the presence of domain 6303 suggest that Vir may be a member of a new class of splice regulators, such as IRE1P, a transmembrane protein that acts as an unconventional splice factor in the unfolded protein response pathway in yeast. However, no sequence homology exists between Vir and IRE1P. The characteristics of Vir led to the speculation that the protein is located in the nuclear membrane where it may mediate mRNA transport. If this were correct, this process could be severely disrupted in strong vir mutants, with mRNAs of vital genes being affected, which would cause sex-unspecific lethality. However, even presumably amorphic alleles are not cell lethal in genetic mosaics (Hilfiker, 1995). In contrast, null alleles of snf, which encodes a component of the general splicing machinery, are incompatible with cell survival. Unlike the strong alleles of vir, the female-specific alleles (vir1ts and vir2f) may reduce effectiveness of the nuclear transportation system such that dose-sensitive processes begin to fail. Autoregulation of Sxl is indeed a sensitive system. Even mutations in aspartyl- as well as in tryptophanyl tRNA synthetase of Drosophila can act as maternal modifiers and disrupt the autoregulation of Sxl. However, the HIS-tagged Vir fusion protein is found in the nucleoplasm of Schneider cells and not in the membrane. This finding does not exclude the possibility of membrane insertion because expression might not be physiological in this experiment and staining from excess protein in the nucleoplasm could mask staining at the periphery. In addition, Vir could be localized to the nucleoplasm and also to the membrane in wild type. These two possibilities are compatible with the speculation about a function in nuclear transport (Niessen, 2001).
In summary, vir emerges as a gene that functions in several developmental pathways: sex determination, dosage compensation and vital processes. It seems to achieve this by regulating Sxl and tra, and presumably msl-2, as well as yet unknown target genes required for vital functions in both sexes. The common molecular mechanism by which vir performs these various functions may be its involvement in the process of splicing whereby the splicing of Sxl, and to a lesser extent of tra and msl-2, appears particularly sensitive to malfunction of vir (Hilfiker, 1995).
A 7.7-kb EcoRI subfragment of a rescue construct was used as a probe to identify vir transcripts and to isolate cDNAs. This probe detected two transcripts on a Northern blot. Three cDNAs were obtained (cVir1-3), which are all colinear and correspond to the larger 6-kb transcript. cVir-1 and cVir-2 span the region encompassing the deletions associated with vir22 and vir23. The 6-kb transcript is encoded by vir.
Three small introns were mapped by comparing the vir cDNAs with the genomic sequence. Two additional introns located upstream of cVir-1 were determined by RT-PCR. There is a long open reading frame of 5562 nucleotides (nt) starting from an ATG codon 66 nt upstream of intron 1. This site (CACAACAUG) has a good match with the consensus for Drosophila translation initiation and is the first start codon in the vir ORF. The transcription start site must lie not more then 50 nt upstream of the ATG start codon because RT-PCR with primers further upstream did not yield amplification products indicating that this region is not present as RNA. If this is correct, the vir transcript contains a small leader while the trailer consists of 204 nt and has no consensus (AATAAA) polyadenylation signal. Nevertheless, all the isolated vir cDNAs had the same 3' end and contained poly (A) tails (Niessen, 2001).
Vir has a calculated molecular weight of 210 kD. A transmembrane domain is predicted at the N terminus of Vir based on the TM-Predict program. Furthermore, Vir contains a consensus nuclear localization signal (NLS) and several PEST domains, which predict that the protein is targeted to the nucleus and has a short half-life in vivo. Amino acids 779 through 805 and two other regions have a high probability of forming coiled coils. In addition, Vir contains a stretch of 144 aa that show homology to domain 6303 of the ProDom database. Domain 6303 is defined by proteins that interact with nucleic acids such as helicases, hnRNPs, or translation initiation factors. Database searches with the conceptual amino acid sequence of Vir retrieved a human protein of 1795 amino acids. An alignment between Vir and this protein reveals that the two sequences have 28% identical and ~40% similar residues. The human protein contains the putative transmembrane domain found in Vir. This points to the importance of this stretch of amino acids for the function of both proteins (Niessen, 2001).
date revised: 25 March 2001
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