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EDAS, a database of alternatively spliced human genes, contains data on the alignment of proteins, mRNAs, and EST. It contains information on all exons and introns observed, as well as elementary alternatives formed from them. The database makes it possible to filter the output data by changing the cut-off threshold by the significance level. The database is accessible at http://www.gene-bee.msu.ru/edas/.
Of the 10 viral genes known to be expressed during Epstein-Barr virus (EBV) latency, six encode nuclear antigens (EBNAs), all of which are expressed from a long primary transcript by means of alternative splicing and alternative polyadenylation sites. The primary transcript is generated by either of two promoters which operate in a mutually exclusive fashion in different clonal cell lines. All mRNAs from either promoter have exons in common from the BamHI W viral genomic fragment (the major internal repeat, IR1) which encode the N-terminal portion of one of the nuclear antigens (EBNA 4). In addition to the coding regions for EBNA 4, EBNA mRNAs encode another EBNA (i.e. EBNA 1, 2, 3A, 3B or 3C) downstream. We show that alternative splicing determines whether the translation initiation codon for EBNA 4 is present or absent, thus permitting the generation of mRNAs in which the first translation initiation codon is either that for the EBNA 4 gene or for the other EBNA gene encoded downstream. This mechanism presumably ensures efficient translation of all the EBNA genes.
Alternative mRNA splicing directed by SR proteins and the splicing regulators TRA and TRA2 is an essential feature of Drosophila sex determination. These factors are highly phosphorylated, but the role of their phosphorylation in vivo is unclear. We show that mutations in the Drosophila LAMMER kinase, Doa, alter sexual differentiation and interact synergistically with tra and tra2 mutations. Doa mutations disrupt sex-specific splicing of doublesex pre-mRNA, a key regulator of sex determination, by affecting the phosphorylation of one or more proteins in the female-specific splicing enhancer complex. Examination of pre-mRNAs regulated similarly to dsx shows that the requirement for Doa is substrate specific. These results demonstrate that a SR protein kinase plays a specific role in developmentally regulated alternative splicing.
We have analyzed nearly 2,000 myosin heavy chain gene (Myh) clones representing over 30 different transcripts from seven of eight striated muscle Myh genes expressed in mouse. We also report the transcriptional start sites (TSS) for the mouse developmental Myh genes. The data reveal a previously unknown diversity of TSSs and 5'-end alternative splicing in these transcripts. The cardiac Myh6 gene had two major TSSs. Use of the major downstream site led to an alternatively spliced second exon. Each of the other Myh genes had one major TATA-directed TSS and one or more minor alternative TSSs, some associated with alternative splicing. The minor transcripts were associated with polysomes and their spatial-temporal expression largely mirrored that of the major transcripts in wild-type, Myh1 null, Myh4 null, injured, and uninjured muscle, except that one form of Myh7, detected in heart, was not detected in diaphragm, and the ratio of the two major Myh6 transcripts varied in some circumstances. These findings indicate that alternative TSS usage and alternative splicing in the 5'-UTR are a general feature of murine Myh gene expression and that Myh gene regulation is more complex than previously appreciated.
The adaptive radiation of cichlid fishes in the lakes of East Africa is a prime example of speciation. The choice of cichlid mates on the basis of a variety of coloration represents a potential basis for speciation that led to adaptive radiation. Here, we characterize the cichlid homolog of the zebrafish hagoromo (hag) gene that was recently cloned and characterized from a pigmentation mutant. Although only one hag mRNA was reported in zebrafish, cichlids express nine different hag mRNAs resulting from alternative splicing. The hag mRNAs are expressed between the myotome and the epidermis where pigment cells are located, suggesting the cichlid hag gene is involved in pigmentation. The hag mRNA splicing pattern does not fluctuate among individuals from each of two species, suggesting that alternative splice site choice is fixed within species. Furthermore, cichlids in lineages that underwent explosive speciation expressed a greater variety of hag mRNAs than those in lineages that did not undergo such a degree of speciation, suggesting that species in the explosively speciated lineage acquired a complex regulatory mechanism of alternative splicing over a very short evolutionary period. Here, we provide an example in which alternative splicing may play a role in mate choice, leading to cichlid speciation through diversification of gene function by production of multiple mRNAs from a single gene.
PURPOSE: The cyclic nucleotide gated cation channel subunit, CNGA3, (also known as the cone alpha CNG subunit), plays a vital role in signal transduction of human cone cells. Owing to its well established role in vision, the human CNGA3 isoform studied thus far was cloned from retinal tissue. However, non-human homologs of CNGA3 have been cloned from a variety of other tissues including kidney, heart, pineal gland, adrenal gland and testes. Among these, alternative splice forms of CNGA3 have been identified. The objective of this study was to explore alternative splice forms of human CNGA3 and determine their distribution among various human tissues. METHODS: RT-PCR was used to amplify full length open reading frames of CNGA3 from human testes RNA and to detect and distinguish among splice forms in 23 tissues. DNA sequencing was used to characterize full length splice forms and to confirm the identity of RT-PCR products from a number of tissues. RESULTS: Two new full length alternatively spliced forms of hCNGA3 (referred to as Variant 2 and 3) were isolated. These splice variants are different from the cone hCNGA3 (Variant 1) in that they lack exon 5. In addition, they differ from each other in that Variant 3 contains an extra exon that originates from the intron preceding exon 4. We demonstrate that CNGA3 transcripts are detectable in all 23 human tissues examined. In all tissues, except retina, Variant 2 specific PCR products had the brightest band intensity. In retina, the band from Variant 1 (containing exon 5) was most intense. In fact, among all tissues examined, Variant 1 can only be detected strongly in the retina. CONCLUSIONS: The extensive distribution of CNGA3 and the tissue specific expression of alternative splice forms indicate widespread and diverse roles for CNGA3. The unique expression of Variant 1 in the retina implies a significance to the amino acids encoded by exon 5 that may be necessary for the function of CNGA3 in human cone cells.
Alternatively spliced tissue factor (asTF) has recently been discovered as a soluble form of tissue factor (TF), which circulates in blood and exhibits procoagulant activity. This soluble TF variant expanded the concept of circulating TF by a further element. Up to 30% of the TF antigen found in circulating blood was proposed to be derived from alternative splicing. We showed that cytokines induced the expression of asTF and the release from endothelial cells. The use of plasma asTF as a clinical marker for an inflammation-associated dysregulated hemostasis may therefore be a novel approach in predicting the patients' prognosis. This review covers the latest findings in the field of soluble TF focusing on asTF and its potential role besides the one in coagulation.
HLA class II antigens are highly polymorphic cell-surface proteins involved in initiation and regulation of the immune response. Allelic sequence variation primarily affects the structure of the first external domains of alpha and beta component chains. Here we provide evidence for other types of allelic polymorphism for the genes encoding these chains. Sequences of two cDNA clones corresponding to HLA-DQB mRNAs from an HLA-homozygous cell line exhibit both alternative splicing and read-through of polyadenylylation. Furthermore, alternative splicing that deletes the transmembrane exon is associated with only a subset of HLA-DQB alleles, while the polyadenylylation-site read-through is found in a larger subset. This suggest that polymorphic cis-acting elements within the HLA-DQB gene control both processing steps. Proteins, presumably encoded by alternatively spliced mRNAs lacking transmembrane exons, are immunoprecipitated with a monomorphic monoclonal antibody directed against HLA-DQ. These proteins are found in supernatants of cultured cell lines for which secretion is predicted, but not in those of cell lines that do not contain alternatively spliced mRNAs.
OBJECTIVE: To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing. METHODS: According to the sequence of human ESTs which are highly homologous to hTCP11a, primers for PCR were synthesized. Then, the amplified fragments were cloned and sequenced; some methods including BLAST, ClustalW and RT-PCR were used for genomic analysis, study of alternative splicing and gene expression among multiple tissues and different testis tissues. RESULTS: A novel isoform of hTCP11 gene was isolated. It encodes a 440 amino acid protein that is highly homologous to the mouse 566 amino acid protein which is important to sperm function because it encodes the receptor for fertilization promoting peptide (FPP). Among TCP11a, TCP11b and TCP11c, the complicated alternative splicing was found. RT-PCR analysis of RNA extracted from human tissues revealed that the gene is only expressed in fertile adult testes, but not in azoospermic patient testes, fetal testes or other human tissues. CONCLUSION: Our results along with the mouse Tcp-11 function suggest that the isoforms of TCP11 gene play important roles in sperm function and fertility.
We cloned genomic DNA corresponding to the Drosophila virilis homologue of para, a gene encoding a sodium channel alpha-subunit, and obtained many partial cDNA clones from embryos and adults. Para protein has been well conserved, and the optional elements at six different sites of alternative splicing in D. melanogaster are present in D. virilis, in addition to one new optional exon. Among 31 different splice-types observed in D. virilis, the stage-specific pattern of alternative splicing seen in D. melanogaster is also conserved. Comparison of genomic DNA sequence revealed three aspects that vary between alternatively and constitutively used exon sequences. Sixteen short blocks (10-75 bp), the only recognizably conserved intron sequence, were disproportionately associated with alternatively used splice sites. Silent site substitutions were found much less frequently in alternative than constitutive exon elements, and the degree of match to the Drosophila splice site consensus tended to be lower at less frequently selected alternative splice junctions. This study shows that the developmentally regulated variability of para products is highly conserved and therefore likely to be of functional significance and suggests that a variety of different sequence-dependent mechanisms may regulate this pattern of alternative splicing.
The insulin receptor exists in two isoforms that result from alternative splicing of exon 11 in the primary transcript. The two isoforms have different affinities for insulin and insulin-like growth factors, and mediate different biological actions by different intracellular pathways. Differential expression of the two isoforms not only might contribute to insulin resistance in type 2 diabetes mellitus, but also play an important role in the pathogenesis of cancer. Alternative splicing can be regulated by metabolic and hormonal factors, such as hyperinsulinemia and hyperglycemia, but the precise mechanism is unknown. It has also been demonstrated that there are certain important nucleotide sequences in insulin receptor gene that can influence alternative splicing.
Telomerase activity is generally considered to be necessary for cancer cells to avoid senescence. The expression of human telomerase reverse transcriptase (hTERT) is believed to be a rate-limiting step in telomerase activation. Recently, it has been proposed that the alternative splicing of hTERT is also involved in regulation of telomerase activity. However, the regulatory mechanism of telomerase in cancer cells has not been thoroughly investigated. To clarify it in lung cancer cells, we measured the expression of the hTERT transcript, analyzed its alternative splicing by RT-PCR, and compared it with telomerase activity and telomere length. The expression of the hTERT transcript was positively correlated with telomerase activity in lung cancer cells. Cancer cells with high telomerase activity contained 4 splicing variants of hTERT, and the full-length variant was 31.3-54.2% of the total transcripts. Cells of the TKB-20 cell line, which has extremely low telomerase activity, showed a different splicing pattern of hTERT in addition to low expression. The functional full-length variant was scarcely detected in TKB-20 cells, suggesting that the telomerase activity was repressed by alternative splicing of hTERT. Telomere length was not necessarily correlated with telomerase activity or hTERT expression in lung cancer cells. Cells of the TKB-4 cell line that also showed relatively low telomerase activity (as TKB-20 cells) had long telomeres. In conclusion, hTERT expression is regulated at both the transcriptional and post-transcriptional levels in lung cancer cells, and the alternative splicing of hTERT is involved in the control of telomerase activity.
The human CD44 gene undergoes extensive alternative splicing of multiple variable exons positioned in a cassette in the middle of the gene. Expression of alternative exons is often restricted to certain tissues and could be associated with tumor progression and metastasis of several human malignancies, including breast cancer. Exon v4 contains multiple copies of a C/A-rich exon enhancer sequence required for optimal inclusion of the exon and binding to the nucleic acid-binding proteins YB-1 and human Tra2-beta1. Here, we show that hTra2-beta1, a member of the extended family of serine/arginine-rich (SR) splicing factors, enhances the in vivo inclusion of CD44 exons v4 and v5. It increased inclusion of exons v4 and v5 and acted synergistically with YB-1. Activation required the C/A-rich enhancer within exon v4. Several other SR proteins had none or only a slight effect on CD44 exon inclusion. In contrast, SC35 inhibited exon usage and antagonized the effects of Tra2 or YB-1. In a matched pair analysis of human breast cancers and their corresponding nonpathologic tissue controls, we found a significant induction of Tra2-beta1 in invasive breast cancer, both on the RNA and protein levels. Together with our functional data, these results suggest an important role for Tra2-beta1 in breast cancer. Induction of this splicing factor might be responsible for splicing of CD44 isoforms associated with tumor progression and metastasis.
Several mechanisms were used in determination of the development of the male or female of vertebrates. The genes for determination of sequential hermaphrodite sex are unknown. Here, we reported cloning, alternative splicing, and expression patterns of the CYP17 gene of the rice field eel, a teleost fish with a characteristic of nature sex reversal. The CYP17 gene of the rice field eel was clustered into the CYP17 gene group of all the other vertebrates, especially into the fish subgroup. Four isoforms of the CYP17 were generated in gonads by alternative splicing and polyadenylation. Alternative splicing events of all these isoforms occurred in 3(') regions, which encoded three different sizes (517, 512, and 159aa) of proteins. RT-PCR results indicate specific expression in gonads of these isoforms. Northern blot analysis shows that expression patterns of the CYP17 (dominantly expressed in testis, less in ovary, and the least in ovotestis) are consistent with the sex reversal process of the rice field eel. In situ hybridization further shows its specific expression in germinal lamellae, the gonadal epithelium of the gonads. These findings indicate that CYP17 is differentially regulated in a sex- and developmentally specific manner, suggesting that the CYP17 potentially has important roles in gonad differentiation during sex reversal of the rice field eel.
The p53-inducible gene 3 (PIG3) is a transcriptional target of the tumor suppressor protein p53 and is thought to play a role in apoptosis. In this report, we identify a novel alternatively spliced product from the PIG3 gene that we call PIG3AS (PIG3 alternative splice). PIG3AS results from alternative pre-mRNA splicing that skips exon 4 of the five exons included in the PIG3 transcript. The resulting protein product shares its first 206 amino acids with PIG3 but has a unique 42-amino acid C terminus. In unstressed cells and after most DNA damage conditions that induce transcription from the PIG3 gene, production of the PIG3 transcript dominates. However, in response to UV light, pre-mRNA splicing shifts dramatically in favor of PIG3AS. Unlike the PIG3 protein, the PIG3AS protein is rapidly degraded with a short half-life and is stabilized by proteasome inhibition. Our results illustrate the first example of an endogenous, UV-inducible, alternative splicing event and that control of the splicing machinery is involved in the cellular DNA damage response. They also suggest that rapid proteolytic degradation represents a cellular mechanism for uncoupling p53 activity from PIG3 gene activation that is independent of promoter selectivity.
BACKGROUND: Four genes designated as PTPRK (PTPkappa), PTPRL/U (PCP-2), PTPRM (PTPmu) and PTPRT (PTPrho) code for a subfamily (type R2B) of receptor protein tyrosine phosphatases (RPTPs) uniquely characterized by the presence of an N-terminal MAM domain. These transmembrane molecules have been implicated in homophilic cell adhesion. In the human, the PTPRK gene is located on chromosome 6, PTPRL/U on 1, PTPRM on 18 and PTPRT on 20. In the mouse, the four genes ptprk, ptprl, ptprm and ptprt are located in syntenic regions of chromosomes 10, 4, 17 and 2, respectively. RESULTS: The genomic organization of murine R2B RPTP genes is described. The four genes varied greatly in size ranging from approximately 64 kb to approximately 1 Mb, primarily due to proportional differences in intron lengths. Although there were also minor variations in exon length, the number of exons and the phases of exon/intron junctions were highly conserved. In situ hybridization with digoxigenin-labeled cRNA probes was used to localize each of the four R2B transcripts to specific cell types within the murine central nervous system. Phylogenetic analysis of complete sequences indicated that PTPrho and PTPmu were most closely related, followed by PTPkappa. The most distant family member was PCP-2. Alignment of RPTP polypeptide sequences predicted putative alternatively spliced exons. PCR experiments revealed that five of these exons were alternatively spliced, and that each of the four phosphatases incorporated them differently. The greatest variability in genomic organization and the majority of alternatively spliced exons were observed in the juxtamembrane domain, a region critical for the regulation of signal transduction. CONCLUSIONS: Comparison of the four R2B RPTP genes revealed virtually identical principles of genomic organization, despite great disparities in gene size due to variations in intron length. Although subtle differences in exon length were also observed, it is likely that functional differences among these genes arise from the specific combinations of exons generated by alternative splicing.
Using in vitro amplification of cDNA by the polymerase chain reaction, we analyzed alternatively spliced events of cystic fibrosis transmembrane conductance regulator gene in lymphoblastoid cells. Ten alternatively spliced transcripts were identified using analysis of 6 overlapping segments of amplified cDNA, 4 of which have not been described previously. These include transcripts lacking exon 16, 17b, 22 and a transcript resulting from the use of a cryptic acceptor and donor splice sites. Moreover, in 2 cystic fibrosis (CF) patients bearing nonsense mutations E60X or W1282X, we observed that nonsense mutations are associated with an alteration of splice site selection in vivo resulting in exon skipping of constitutive exons or in the use of cryptic splice sites. In addition, even though lymphoblastoid cells are not the relevant tissue to address the question of the relationship between clinical respiratory phenotype and genotype, our results concerning adult CF patients (delta F508/ delta F508) suggest that individual-specific RNA splicing patterns could influence the severity of the CF pulmonary disease. If this phenomenon of alternative splicing events proves to be significant in CF and to be a common feature of disease genes, the study of RNA splicing could become an important tool for the analysis of the genotype-phenotype relationship in many inherited disorders.
The sequences of the human and bovine genes for the phosphate carrier from the inner membranes of mitochondria have been determined. The genes have similar structures and each is divided into nine exons. In both genes, two exons, named IIIA and IIIB, are closely related, and they appear to the alternatively spliced. The human exon IIIB sequence is found in a published human heart cDNA sequence, and bovine exon IIIA forms part of a published bovine heart cDNA sequence. By further examination of the human heart cDNA library, sequences arising from both alternatively spliced forms of the phosphate carrier have been characterized. Both forms were also found in several bovine tissues, but the ratios of expression of the two forms varied. The form containing exon IIIA was expressed most highly in bovine heart and liver, less highly in brain and kidney, and only in low amounts in lung. The opposite hierarchy was found for the form containing exon IIIB; it was most highly expressed in lung and least in heart and liver. The alternative splicing mechanism affects amino acids 4-45 of the mature phosphate carrier protein, which is believed to form one of six transmembrane segments of the phosphate carrier and to emerge into a large extramembranous loop. The alternative splicing mechanism changes 13 and 11 amino acids in the human and bovine carrier proteins, respectively. As the function of this region of the phosphate carrier is not known, the effects of the changes on carrier function are not understood at present.