Gene name - sans fille
Synonyms - simply not fertile, D25, U1A snRNP
Cytological map position - 4F1-4F2
Symbol - snf
FlyBase ID: FBgn0003449
Genetic map position - 1-11.7
Classification - RRM motif
Cellular location - nuclear and cytoplasmic
From the standpoint of developmental biology, the alternative splicing of exons is intimately connected with the spice of life: variety. This overview will discuss the complex, variety-producing process known as alternative splicing, and aspects of the process involving the gene snf.
In the most general terms, splicing is the removal of an intron, a specified portion of the nucleotide chain, from between two flanking exons (portions of the pre-messenger RNA that code for proteins). The process is regulated by certain proteins called splice factors, and depends upon the successful construction of splice machinery, in particular a structure known as a spliceosome, a multiprotein complex that is assembled step by step during the splicing process. Alternative splicing generates alternative messenger RNA species, depending on the pattern of intron removal. For example, by alternative splicing in which exons are skipped, unwanted exons can be excluded altogether from mRNA species coding for a particular polypeptide chain, thus generating a functionally alternative protein.
By way of an oversimplified analogy, imagine a nucleotide chain of people (introns and exons) holding hands. As some people leave the line, those remaining will join hands (or be spliced) with new neighbors on one side or the other or both. Depending on the specific intron(s) removed, the exons will find themselves adjacent to different exon neighbors. This brings about a re-arrangement of the nucleic acid pre-splice sequence; from this new sequence, depending on how and where the exons have been shuffled, an altered code is now in place, providing different proteins from a unified pre-splice, pre-messenger RNA. These changes in coding sequences and hence, changes in the proteins produced, can mean the difference between an embryo developing into a male or a female; cell fate rarely gets more basic than this.
The splice machinery of the cell is found in the nucleoplasm of the cell. The idea that the nucleoplasm, as distinct from the DNA (the bearer of the genetic code of the cell), is a determiner of cell fate is an idea whose time has come. By generating alternative versions of proteins, for examples, alternative versions of splice factors or transcription factors, the nucleoplasm-based splice machinery regulates cell fate. For a more detailed version of how the nucleoplasm constructs the spliceosome, the protein machinery that carries out splicing, see Transformer 2.
sans fille is now known as a splice factor. It has revealed itself to interested investigators only very slowly. Its initial identity, established from mutation studies, was as a sex determination gene (Oliver, 1988). Only when snf was cloned, and its sequence identified, was its even more basic nature revealed. SNF is similar in sequence and functionally identical to U1A snRNP (small nuclear ribonucleoprotein), a protein of major importance in splicing. SNF's role in alternative splicing places it directly in the sex determination hierarchy, as a factor involved in Sex lethal's autoregulation (Flickinger, 1994).
How does snf function in sex determination? snf or U1A snRNP as it is usually called when refering to it as a splice factor, is one of the few major proteins responsive to factors involved in alternative splicing. snf's role in germline sex determination was discovered directly from the genetic analysis of a single allele, snf1621. Females homozygous for snf1621 are sterile. In the mutant neither the oocyte nor the nurse cells differentiate. Instead the germinal cells continue to divide, resulting in the formation of ovarian tumors. A similar phenotype is seen in fused, ovo, and otu mutants. The similarity of snf and Sxl mutant phenotypes in the germline, and the fact that they interact genetically, suggests that they disrupt the same process, germline sex determination (Salz, 1992 and references).
In contrast to snf's role in germline sex determination, its role in somatic sex differentiation can only be inferred by an unusual genetic interaction between mutations at the snf and Sxl loci. Although doubly heterozygous females have reduced viability, many surviving females show signs of sex transformations. snf is required for the activation of Sex-lethal in both the germline and the soma (Salz, 1992).
Genetic evidence alone was insufficient to pinpoint the role of snf in sex determination. A clue was provided in a study of SXL ovarian expression in snf1621 mutants. The splicing pattern of SXL transcripts in ovaries mutant for snf1621 shows substantial levels of male-spliced SXL mRNA. In wild type females, three major classes of SXL mRNAs are observed. In snf1621 mutants, one of these three classes is not detectable and a second is reduced (Salz, 1992). The germ cells populating snf1621 mutants have little or no SXL protein. This result is consistent with the idea that regulation of SXL splicing is impaired in germ cells mutant for snf1621 (Salz, 1992 and Bopp, 1993).
Further analysis revealed aberrant Sex-lethal regulation in snf mutants in late embryogenesis. The role of snf somatic sex determination is only evident when the probability of Sxl activation is reduced. In this case, embryonic female-specific lethality is caused by a lack of maternal snf function. By examining both the RNA and protein expression pattern of Sxl in embryos mutant for snf, it is found that Sxl is not stably activated. Although both the early female-specific pattern of Sxl mRNA and the early SXL protein expression appear normal, there is little or no accumulation of the female-specific spliced form of the late transcripts, and consequently little or no SXL protein is found late in embryogenesis (Albrecht, 1993).
Cloning of snf reveals a protein with two RNA recognition motifs (RRMs); both are similar to two closely related small nuclear ribonuclear proteins (snRNPs): U1A and U2B". These two RNA-binding proteins bind different RNAs. U1a binds U1 snRNA, and U2B" binds U2 snRNA. Both proteins and their bound RNAs are intimately involved is splicing. In many respects, SNF resembles U2B" more closely than U1A. Not only does SNF have a similar overall structure and molecular weight to U2B" but it also contains a 5-amino-acid motif within the amino-terminal RRM that is identical to that found in U2B". In the human protein, this region has been shown to be necessary for the U1A protein to discriminate between U1 and U2 RNA. Functional studies demonstrate the true identity of SNF as a U1 snRNP. In both Drosophila and human cell extracts, an epitope-tagged SNF protein immunoprecipitates from U1 small nuclear RNA (snRNA) but not U2 snRNA (Harper, 1992).
How does SNF relate to alternative splicing? The splicing of SXL pre-mRNA is autoregulated by Sex lethal protein. SNF cooperates with SXL to block utilization of the male-specific exon of the SXL pre-mRNA, suggesting a model in which the SXL protein blocks spliceosome assembly by forming a non-productive snRNA/SXL complex. By blocking the male-specific exon, SXL assures that only the female mRNA, capable of coding for a complete SXL protein, is made in females.
Initially, SXL protein binds to intron sequences far distant from the male exon (exon 3), and by so doing, it blocks the utilization of male exon splice sites. SXL proteins then bind to the poly U tracts in the introns upstream and downstream of the SXL male exon. SXL proteins in the intron then make contact with the U1 snRNPs associated with the male exon splice junctions, presumably via protein-protein interactions with SNF. These contacts would prevent the snRNPs at the male exon splice junctions from participating in subsequent splicing steps (Saltz, 1996 and Deshpande, 1996).
The role of U1 snRNP in alternative splicing is not confined to SXL pre-mRNA. Members of the SR family of proteins can collaborate with U1 snRNP in the recognition of 5' splice sites in pre-messenger RNAs. Individual SR proteins have distinct abilities to promote interaction of U1 snRNP with alternative 5' splice junctions. (See snf Evolutionary homologs for additional information). In Drosophila, the proteins Transformer and Transformer 2 have conserved SR domains and function in an identical fashion to other SR proteins in influencing alternative splicing of the Doublesex pre-mRNA. SR proteins act as a bridge between U1, associated with the 5' splice site and U2AF65/U2AF35 (see U2 small nuclear riboprotein auxiliary factor 50) assembled at the 3' splice site. The highly conserved SR protein RBP1 targets sequences within the DSX repeat region that are required for the efficient splicing of DSX pre-mRNA (Heinrichs, 1995). Essentially SR proteins are involved in the assembly of the spliceosome. It is thought that SR proteins can regulate alternative splicing in many pre-mRNAs.
U1 snRNP is really a multiprotein complex, of which the A protein (Sans fille) is but one component. The A protein makes direct physical contact with the U1 snRNA, the RNA component of the snRNP. This RNA component is involved in direct recognition of the conserved sequence of the 5' splice sites of pre-mRNA introns. The A component is also known to interact with proteins involved in mRNA polyadenylation. The U1-70K subunit is known to directly interact with SR proteins in vertebrates. U1-C lacks an RNA binding domain and does not appear to bind directly to U1 snRNA. However, at the amino terminal end protein C contains a zinc finger-like structure of the CC-HH type. Several lines of evidence indicate that the zinc finger-like structure is essential for the binding of protein C to U1 snRNP particles. U1 snRNP is known to make physical contact with the cap-binding complex (CBC), which specifically recognizes the monomethyl guanosine cap structure carried by RNA polymerase II transcripts. CBC is required for the efficient recognition of the 5' splice site by U1 snRNP during formation of a complex on the first intron of pre-mRNA. The number and complexity of known U1 snRNP interactions will continue to rise as more information is acquired on spliceosome biology.
Exons - 2
Bases in 3' UTR - 77
The predicted protein contains two RNA recognition motifs (RRMs), one at the amino terminus and the other at the carboxyl terminus. This protein shows extensive overall similarity to two closely related human snRNP proteins, U1A and U2B". The amino acid identity between SNF and these two human proteins is respectively 72% and 70%. Considering only the amino terminal RRM domain, which is responsible for the snRNA-binding specificity of U1A and U2B", the identity is even stronger: 82.5% with U1A and 79% for U2B". SNF protein is identical to a previously cloned Drosophila gene, D25 (Harper, 1992), reported to have functional similarity to U1A (Flickinger, 1994).
A recombinant Drosophila RNA binding protein has been characterized, D25 (now known as Sans fille), by virtue of its antigenic relationship to mammalian U1 and U2 small nuclear ribonucleoprotein (U snRNP) proteins. Sequence analysis reveals that D25 bears strong similarity to both the human U1 snRNP-A (U1-A) and U2 snRNP-B" (U2-B") proteins. However, at residues known to be critical for the RNA binding specificities of U1-A and U2-B" D25 sequence is more similar to U2-B". Using direct RNA binding assays D25 selects U1 RNA from either HeLa or Drosophila Kc cell total RNA. Furthermore, D25 binds U1 RNA when transfected into mammalian cells. Thus, D25 appears to be a Drosophila homolog of the mammalian U1-A protein, despite its sequence similarity to U2-B" (Harper, 1992).
date revised: 12 Mar 97
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