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Query: UNIPROT:P06889 (Mol)
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Sex determination in Drosophila melanogaster is regulated by a cascade of splicing factors which direct the sex-specific expression of gene products needed for male and female differentiation. The splicing factor TRA-2 affects sex-specific splicing of multiple pre-mRNAs involved in sexual differentiation. The tra-2 gene itself expresses a complex set of mRNAs generated through alternative processing that collectively encode three distinct protein isoforms. The expression of these isoforms differs in the soma and germ line. In the male germ line the ratio of two isoforms present is governed by autoregulation of splicing. However, the functional significance of multiple TRA-2 isoforms has remained uncertain. Here we have examined whether the structure, function, and regulation of tra-2 are conserved in Drosophila virilis, a species diverged from D. melanogaster by over 60 million years. We find that the D. virilis homolog of tra-2 produces alternatively spliced RNAs encoding a set of protein isoforms analogous to those found in D. melanogaster. When introduced into the genome of D. melanogaster, this homolog can functionally replace the endogenous tra-2 gene for both normal female sexual differentiation and spermatogenesis. Examination of alternative mRNAs produced in D. virilis testes suggests that germ line-specific autoregulation of tra-2 function is accomplished by a strategy similar to that used in D. melanogaster. The similarity in structure and function of the tra-2 genes in these divergent Drosophila species supports the idea that sexual differentiation in D. melanogaster and D. virilis is accomplished under the control of similar regulatory pathways.
Mol Cell Biol 1997 May
PMID:Evolutionary conservation of regulatory strategies for the sex determination factor transformer-2. 911 63

We have examined the effect of RCC1 function on the nuclear organization of pre-mRNA splicing factors and poly(A)+ RNA in the tsBN2 cells, a RCC1 temperature-sensitive mutant cell line. We have found that at 4-6 h after shifting cells from the permissive temperature (32.5 degrees C) to the restrictive temperature (39.5 degrees C), both small nuclear ribonucleoprotein particles and a general splicing factor SC35 reorganized into 4-10 large round clusters in the nucleus, as compared with the typical speckled distribution seen in cells at the permissive temperature. In situ hybridization to poly(A)+ RNA resulted in a similar pattern. Examination by double labeling demonstrated that the redistribution of splicing factors coincides with that of poly(A)+ RNA. Such changes in the nuclear organization of splicing factors and poly(A)+ RNA were not the result of the temperature shift or of chromatin condensation. Cellular transcription was not significantly altered in these cells and extracts made from both the permissive and restrictive temperature were splicing competent. Electron microscopic examination demonstrated that the large clusters containing both splicing factors and poly(A)+ RNA were fused interchromatin granule clusters. In addition, small electron-dense dot-like structures measuring approximately 80 nm in diameter were also observed, most of which are accumulated in enlarged interchromatin granule clusters in the nucleoplasm of RCC1- cells. In spite of the significant changes observed in the nucleoplasm, relatively little alteration was observed in nucleolar structure by both light and electron microscopic examination. The above observations suggest that the RCC1 protein directly or indirectly regulates the organization of splicing components and poly(A)+ RNA in the cell nucleus and that RCC1 may play a role in nuclear organization.
Mol Biol Cell 1997 Jun
PMID:RCC1 and nuclear organization. 920 22

Galectins are a family of beta-galactoside-binding proteins that contain characteristic amino acid sequences in the carbohydrate recognition domain (CRD) of the polypeptide. The polypeptide of galectin-1 contains a single domain, the CRD. The polypeptide of galectin-3 has two domains, a carboxyl-terminal CRD fused onto a proline- and glycine-rich amino-terminal domain. In previous studies, we showed that galectin-3 is a required factor in the splicing of nuclear pre-mRNA, assayed in a cell-free system. We now document that (i) nuclear extracts derived from HeLa cells contain both galectins-1 and -3; (ii) depletion of both galectins from the nuclear extract either by lactose affinity adsorption or by double-antibody adsorption results in a concomitant loss of splicing activity; (iii) depletion of either galectin-1 or galectin-3 by specific antibody adsorption fails to remove all of the splicing activity, and the residual splicing activity is still saccharide inhibitable; (iv) either galectin-1 or galectin-3 alone is sufficient to reconstitute, at least partially, the splicing activity of nuclear extracts depleted of both galectins; and (v) although the carbohydrate recognition domain of galectin-3 (or galectin-1) is sufficient to restore splicing activity to a galectin-depleted nuclear extract, the concentration required for reconstitution is greater than that of the full-length galectin-3 polypeptide. Consistent with these functional results, double-immunofluorescence analyses show that within the nucleus, galectin-3 colocalizes with the speckled structures observed with splicing factor SC35. Similar results are also obtained with galectin-1, although in this case, there are areas of galectin-1 devoid of SC35 and vice versa. Thus, nuclear galectins exhibit functional redundancy in their splicing activity and partition, at least partially, in the nucleoplasm with another known splicing factor.
Mol Cell Biol 1997 Aug
PMID:Evidence for a role for galectin-1 in pre-mRNA splicing. 923 29

Previous studies have demonstrated a role for the fos gene in promoting malignant conversion of mouse skin tumors. In the study reported here, differential display was performed to identify fos- and jun-regulated genes that are differentially expressed during premalignant progression. Total RNA isolated from variants of the papilloma cell line SP-1 transduced with retroviral vectors expressing v-jun and v-fos alone or in tandem was analyzed for the presence of differentially expressed transcripts by using 35 different primer combinations. Differentially expressed clones were rescreened by dot-blot analysis by using cDNA from chemically induced tumors with a high or low risk of malignant conversion. Three differentially displayed fragments were isolated in this analysis. Homology searches indicated that these fragments shared significant homology with the apoptosis inhibitor bcl-2, human alternative splicing factor/splicing factor 2 (ASF/SF2), and a novel gene not present in the GenBank or EMBL databases. In situ hybridization indicated that the expression levels of the bcl-2 homolog increased with malignant potential in chemically derived mouse skin tumors. A similar analysis indicated that expression of the ASF/SF2 homolog was greater in papillomas than in normal skin or in squamous cell carcinomas. Transcripts for this gene were most abundant in the granular layer. The expression pattern of the third differential display fragment was consistent with that of a tumor suppressor gene. This gene was expressed at very high levels in normal skin and benign papillomas but was essentially undetectable in squamous cell carcinomas. Through this approach, we identified known and novel genes that may contribute to malignant progression in epidermal tumors.
Mol Carcinog 1997 Sep
PMID:Identification of differentially expressed genes in chemically induced skin tumors. 932 39

Nuclear RNA splicing occurs in an RNA-protein complex, termed the spliceosome. U4/U6 snRNP is one of four essential small nuclear ribonucleoprotein (snRNP) particles (U1, U2, U5 and U4/U6) present in the spliceosome. U4/U6 snRNP contains two snRNAs (U4 and U6) and a number of proteins. We report here the identification and characterization of two human genes encoding U4/U6-associated splicing factors, Hprp3p and Hprp4p, respectively. Hprp3p is a 77 kDa protein, which is homologous to the Saccharomyces cerevisiae splicing factor Prp3p. Amino acid sequence analysis revealed two putative homologues in Caenorhabditis elegans and Schizosaccharomyces pombe. Polyclonal antibodies against Hprp3p were generated with His-tagged Hprp3p over-produced in Escherichia coli . This splicing factor can co-immunoprecipitate with U4, U6 and U5 snRNAs, suggesting that it is present in the U4/U6.U5 tri-snRNP. Hprp4p is a 58 kDa protein homologous to yeast splicing factor Prp4p. Like yeast Prp4p, the human homologue contains repeats homologous to the beta-subunit of G-proteins. These repeats are called WD repeats because there is a highly conserved dipeptide of tryptophan and aspartic acid present at the end of each repeat. The primary amino acid sequence homology between human Hprp4p and yeast Prp4p led to the discovery of two additional WD repeats in yeast Prp4p. Structural homology between these human and yeast splicing factors and the beta-subunit of G-proteins has been identified by sequence-similarity comparison and analysis of the protein folding by threading. Structural models of Hprp4p and Prp4p with a seven-blade beta-propeller topology have been generated based on the structure of beta-transducin. Hprp3p and Hprp4p have been shown to interact with each other and the first 100 amino acids of Hprp3p are not essential for this interaction. These experiments suggest that both Hprp3p and Hprp4p are components of human spliceosomes.
Hum Mol Genet 1997 Nov
PMID:Identification and characterization of human genes encoding Hprp3p and Hprp4p, interacting components of the spliceosome. 932 76

Both experimental work and surveys of the lengths of internal exons in nature have suggested that vertebrate internal exons require a minimum size of approximately 50 nucleotides for efficient inclusion in mature mRNA. This phenomenon has been ascribed to steric interference between complexes involved in recognition of the splicing signals at the two ends of short internal exons. To determine whether U1 small nuclear ribonucleoprotein, a multicomponent splicing factor that is involved in the first recognition of splice sites, contributes to the lower size limit of vertebrate internal exons, we have taken advantage of our previous observation that U1 small nuclear RNAs (snRNAs) which bind upstream or downstream of the 5' splice site (5'SS) stimulate splicing of the upstream intron. By varying the position of U1 binding relative to the 3'SS, we show that U1-dependent splicing of the upstream intron becomes inefficient when U1 is positioned 48 nucleotides or less downstream of the 3'SS, suggesting a minimal distance between U1 and the 3'SS of approximately 50 nucleotides. This distance corresponds well to the suggested minimum size of internal exons. The results of experiments in which the 3'SS region of the reporter was duplicated suggest an optimal distance of greater than 72 nucleotides. We have also found that inclusion of a 24-nucleotide miniexon is promoted by the binding of U1 to the downstream intron but not by binding to the 5'SS. Our results are discussed in the context of models to explain constitutive splicing of small exons in nature.
Mol Cell Biol 1997 Dec
PMID:U1 small nuclear RNA-promoted exon selection requires a minimal distance between the position of U1 binding and the 3' splice site across the exon. 937 41

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5' and 3' splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.
Mol Cell Biol 1998 Feb
PMID:Sip1, a novel RS domain-containing protein essential for pre-mRNA splicing. 944 63

We report the cloning of the noisette gene (noi), which encodes the Drosophila melanogaster ortholog of a U2 snRNP-associated splicing factor, SF3a60 (SAP61) in humans and PRP9p in Saccharomyces cerevisiae. Antibodies raised against human SF3a60 recognized NOI in flies, showing a nuclear localization in all the stages examined, including the embryo, the dividing cells of imaginal discs, and the larval polyploid nuclei. NOI is expressed in somatic and germinal cells of both male and female gonads. By mobilization of P transposons, we have generated a large number of noi mutations. Complete loss of function resulted in lethality at the end of embryogenesis, without obvious morphological defects. Hypomorphic alleles revealed multiple roles of noi for the survival and differentiation of male germ cells, the differentiation of female germ cells, and the development of several adult structures.
Mol Cell Biol 1998 Apr
PMID:Multiple developmental requirements of noisette, the Drosophila homolog of the U2 snRNP-associated polypeptide SP3a60. 952 55

A genetic screen was devised to identify Saccharomyces cerevisiae splicing factors that are important for the function of the 5' end of U2 snRNA. Six slt (stands for synthetic lethality with U2) mutants were isolated on the basis of synthetic lethality with a U2 snRNA mutation that perturbs the U2-U6 snRNA helix II interaction. SLT11 encodes a new splicing factor and SLT22 encodes a new RNA-dependent ATPase RNA helicase (D. Xu, S. Nouraini, D. Field, S. J. Tang, and J. D. Friesen, Nature 381:709-713, 1996). The remaining four slt mutations are new alleles of previously identified splicing genes: slt15, previously identified as prp17 (slt15/prp17-100), slt16/smd3-1, slt17/slu7-100, and slt21/prp8-21. slt11-1 and slt22-1 are synthetically lethal with mutations in the 3' end of U6 snRNA, a region that affects U2-U6 snRNA helix II; however, slt17/slu7-100 and slt21/prp8-21 are not. This difference suggests that the latter two factors are unlikely to be involved in interactions with U2-U6 snRNA helix II but rather are specific to interactions with U2 snRNA. Pairwise synthetic lethality was observed among slt11-1 (which affects the first step of splicing) and several second-step factors, including slt15/prp17-100, slt17/slu7-100, and prp16-1. Mutations in loop 1 of U5 snRNA, a region that is implicated in the alignment of the two exons, are synthetically lethal with slu4/prp17-2 and slu7-1 (D. Frank, B. Patterson, and C. Guthrie, Mol. Cell. Biol. 12:5179-5205, 1992), as well as with slt11-1, slt15/prp17-100, slt17/slu7-100, and slt21/prp8-21. These same U5 snRNA mutations also interact genetically with certain U2 snRNA mutations that lie in the helix I and helix II regions of the U2-U6 snRNA structure. Our results suggest interactions among U2 snRNA, U5 snRNA, and Slt protein factors that may be responsible for coupling and coordination of the two reactions of pre-mRNA splicing.
Mol Cell Biol 1998 Apr
PMID:Synthetic lethality of yeast slt mutations with U2 small nuclear RNA mutations suggests functional interactions between U2 and U5 snRNPs that are important for both steps of pre-mRNA splicing. 952 78

The yeast protein Prp19p is essential for pre-mRNA splicing and is associated with the spliceosome concurrently with or just after dissociation of U4 small nuclear RNA. In splicing extracts, Prp19p is associated with several other proteins in a large protein complex of unknown function, but at least one of these proteins is also essential for splicing (W.-Y. Tarn, C.-H. Hsu, K.-T. Huang, H.-R. Chen, H.-Y. Kao, K.-R. Lee, and S.-C. Cheng, EMBO J. 13:2421-2431, 1994). To identify proteins in the Prp19p-associated complex, we have isolated trans-acting mutations that exacerbate the phenotypes of conditional alleles of prp19, using the ade2-ade3 sectoring system. A novel splicing factor, Snt309p, was identified through such a screen. Although the SNT309 gene was not essential for growth of Saccharomyces cerevisiae under normal conditions, yeast cells containing a null allele of the SNT309 gene were temperature sensitive and accumulated pre-mRNA at the nonpermissive temperature. Far-Western blot analysis revealed direct interaction between Prp19p and Snt309p. Snt309p was shown to be a component of the Prp19p-associated complex by Western blot analysis. Immunoprecipitation studies demonstrated that Snt309p was also a spliceosomal component and associated with the spliceosome in the same manner as Prp19p during spliceosome assembly. These results suggest that the functions of Prp19p and Snt309p in splicing may require coordinate action of these two proteins.
Mol Cell Biol 1998 Apr
PMID:Snt309p, a component of the Prp19p-associated complex that interacts with Prp19p and associates with the spliceosome simultaneously with or immediately after dissociation of U4 in the same manner as Prp19p. 952 91


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