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PubMed2004

Regulation of the large (approximately 1000 kb) imprinted murine Ube3a antisense transcript by alternative exons upstream of Snurf/Snrpn.

Most cases of Angelman syndrome (AS) result from loss or inactivation of ubiquitin protein ligase 3A (UBE3A), a gene displaying maternal-specific expression in brain. Epigenetic silencing of the paternal UBE3A allele in brain appears to be mediated by a non-coding UBE3A antisense (UBE3A-ATS). In human, UBE3A-ATS extends approximately 450 kb to UBE3A from the small nuclear ribonucleoprotein N (SNURF/SNRPN) promoter region that contains a cis-acting imprinting center (IC). The concept of a single large antisense transcript is difficult to reconcile with the observation that SNURF/SNRPN shows a ubiquitous pattern of expression while the more distal part of UBE3A-ATS, which overlaps UBE3A, is brain specific. To address this problem, we examined murine transcripts initiating from several alternative exons dispersed within a 500 kb region upstream of Snurf/Snrpn. Similar to Ube3a-ATS, these upstream (U) exon-containing transcripts are expressed at neuronal stages of differentiation in a cell culture model of neurogenesis. These findings suggest the novel hypothesis that brain-specific transcription of Ube3a-ATS is regulated by the U exons rather than Snurf/Snrpn exon 1 as previously suggested from human studies. In support of this hypothesis, we describe U-Ube3a-ATS transcripts where U exons are spliced to Ube3a-ATS with the exclusion of Snurf-Snrpn. We also show that the murine U exons have arisen by genomic duplication of segments that include elements of the IC, suggesting that the brain specific silencing of Ube3a is due to multiple alternatively spliced IC-Ube3a-ATS transcripts.

Alleles↗

PubMed2005

Significant expansion of exon-bordering protein domains during animal proteome evolution.

We present evidence of remarkable genome-wide mobility and evolutionary expansion for a class of protein domains whose borders locate close to the borders of their encoding exons. These exon-bordering domains are more numerous and widely distributed in the human genome than other domains. They also co-occur with more diverse domains to form a larger variety of domain architectures in human proteins. A systematic comparison of nine animal genomes from nematodes to mammals revealed that exon-bordering domains expanded faster than other protein domains in both abundance and distribution, as well as the diversity of co-occurring domains and the domain architectures of harboring proteins. Furthermore, exon-bordering domains exhibited a particularly strong preference for class 1-1 intron phase. Our findings suggest that exon-bordering domains were amplified and interchanged within a genome more often and/or more successfully than other domains during evolution, probably the result of extensive exon shuffling and gene duplication events. The diverse biological functions of these domains underscore the important role they play in the expansion and diversification of animal proteomes.

Animals↗

PubMed2005

The splicing regulatory element, UGCAUG, is phylogenetically and spatially conserved in introns that flank tissue-specific alternative exons.

Previous studies have identified UGCAUG as an intron splicing enhancer that is frequently located adjacent to tissue-specific alternative exons in the human genome. Here, we show that UGCAUG is phylogenetically and spatially conserved in introns that flank brain-enriched alternative exons from fish to man. Analysis of sequence from the mouse, rat, dog, chicken and pufferfish genomes revealed a strongly statistically significant association of UGCAUG with the proximal intron region downstream of brain-enriched alternative exons. The number, position and sequence context of intronic UGCAUG elements were highly conserved among mammals and in chicken, but more divergent in fish. Control datasets, including constitutive exons and non-tissue-specific alternative exons, exhibited a much lower incidence of closely linked UGCAUG elements. We propose that the high sequence specificity of the UGCAUG element, and its unique association with tissue-specific alternative exons, mark it as a critical component of splicing switch mechanism(s) designed to activate a limited repertoire of splicing events in cell type-specific patterns. We further speculate that highly conserved UGCAUG-binding protein(s) related to the recently described Fox-1 splicing factor play a critical role in mediating this specificity.

Alternative Splicing↗

PubMed2005

Exonization of AluYa5 in the human ACE gene requires mutations in both 3' and 5' splice sites and is facilitated by a conserved splicing enhancer.

Ancient Alu elements have been shown to be included in mature transcripts by point mutations that improve their 5' or 3' splice sites. We have examined requirements for exonization of a younger, disease-associated AluYa5 in intron 16 of the human ACE gene. A single G>C transversion in position -3 of the new Alu exon was insufficient for Alu exonization and a significant inclusion in mRNA was only observed when improving several potential splice donor sites in the presence of 3' CAG. Since complete Alu exonization was not achieved by optimizing traditional splicing signals, including the branch site, we tested whether auxiliary elements in AluYa5 were required for constitutive inclusion. Exonization was promoted by a SELEX-predicted heptamer in Alu consensus sequence 222-228 and point mutations in highly conserved nucleotides of this heptamer decreased Alu inclusion. In addition, we show that Alu exonization was facilitated by a subset of serine/arginine-rich (SR) proteins through activation of the optimized 3' splice site. Finally, the haplotype- and allele-specific ACE minigenes generated similar splicing patterns in both ACE-expressing and non-expressing cells, suggesting that previously reported allelic association with plasma ACE activity and cardiovascular disease is not attributable to differential splicing of introns 16 and 17.

Alu Elements↗

PubMed2000

Four types of calpastatin isoforms with distinct amino-terminal sequences are specified by alternative first exons and differentially expressed in mouse tissues.

Calpastatin, a specific inhibitor of calpain, consists of a unique N-terminal domain (domain L) and four repetitive protease-inhibitor domains (domains 1-4). The isolated cDNAs from various mammalian species have conspicuous differences in the regions encoding the N-terminal sequences and can be classified into four types. Mouse and bovine calpastatins (Type I and Type II, respectively), which also differ from each other in the uttermost N-terminal regions, possess longer domain L sequences than those of rabbit, pig, and human inhibitors (Type III). A sequence of a shorter isoform, registered in a DNA data bank, starts from a part of domain 2 with a different N-terminal sequence (Type IV). To clarify the source of this molecular diversity, we investigated the entire exon-intron organization of the mouse calpastatin gene. The previously obtained mouse calpastatin cDNA is encoded by as many as 31 exons including the first exon, designated 1xa. Three additional exons specifying the N-terminal sequences of the other types (designated exons 1xb, 1u, and 14t, respectively) were identified in the mouse genomic DNA sequence. While the mRNAs for Types I and III were expressed at high levels in liver, the Type II mRNA was abundant in heart and skeletal muscle and expressed at lower levels in liver, brain and testis. The Type IV mRNA was specifically expressed in testis among the tissues examined. These results suggest that the calpastatin isoforms possessing different N-terminal sequences are generated by alternative transcription initiation from their own promoters and skipping of the mutually exclusive exons.

Alternative Splicing↗

PubMed2003

Neutral substitutions occur at a faster rate in exons than in noncoding DNA in primate genomes.

Point mutation rates in exons (synonymous sites) and noncoding (introns and intergenic) regions are generally assumed to be the same. However, comparative sequence analyses of synonymous substitutions in exons (81 genes) and that of long intergenic fragments (141.3 kbp) of human and chimpanzee genomes reveal a 30%-60% higher mutation rate in exons than in noncoding DNA. We propose a differential CpG content hypothesis to explain this fundamental, and seemingly unintuitive, pattern. We find that the increased exonic rate is the result of the relative overabundance of synonymous sites involved in CpG dinucleotides, as the evolutionary divergence in non-CpG sites is similar in noncoding DNA and synonymous sites of exons. Expectations and predictions of our hypothesis are confirmed in comparisons involving more distantly related species, including human-orangutan, human-baboon, and human-macaque. Our results suggest an underlying mechanism for higher mutation rate in GC-rich genomic regions, predict nonlinear accumulation of mutations in pseudogenes over time, and provide a possible explanation for the observed higher diversity of single nucleotide polymorphisms (SNPs) in the synonymous sites of exons compared to the noncoding regions.

Animals↗

PubMed2003

Intronic sequences flanking alternatively spliced exons are conserved between human and mouse.

Comparison of the sequences of mouse and human genomes revealed a surprising number of nonexonic, nonexpressed conserved sequences, for which no function could be assigned. To study the possible correlation between these conserved intronic sequences and alternative splicing regulation, we developed a method to identify exons that are alternatively spliced in both human and mouse. We compiled two exon sets: one of alternatively spliced conserved exons and another of constitutively spliced conserved exons. We found that 77% of the conserved alternatively spliced exons were flanked on both sides by long conserved intronic sequences. In comparison, only 17% of the conserved constitutively spliced exons were flanked by such conserved intronic sequences. The average length of the conserved intronic sequences was 103 bases in the upstream intron and 94 bases in the downstream intron. The average identity levels in the immediately flanking intronic sequences were 88% and 80% for the upstream and downstream introns, respectively, higher than the conservation levels of 77% that were measured in promoter regions. Our results suggest that the function of many of the intronic sequence blocks that are conserved between human and mouse is the regulation of alternative splicing.

3' Flanking Region↗

PubMed2005

Post-entrapment genome engineering: first exon size does not affect the expression of fusion transcripts generated by gene entrapment.

Gene trap mutagenesis in mouse embryonic stem cells has been widely used for genome-wide studies of mammalian gene function. However, while large numbers of genes can be disrupted, individual mutations may suffer from limitations due to the structure and/or placement of targeting vector. To extend the utility of gene trap mutagenesis, replaceable 3' [or poly(A)] gene trap vectors were developed that permit sequences inserted in individual entrapment clones to be engineered by Cre-mediated recombination. 3' traps incorporating different drug resistance genes could be readily exchanged, simply by selecting for the drug-resistance gene of the replacement vector. By substituting different 3' traps, we show that otherwise identical fusion genes containing a large first exon (804 nt) are not expressed at appreciably lower levels than genes expressing small first exons (384 and 151 nt). Thus, size appears to have less effect on the expression and processing of first exons than has been reported for internal exons. Finally, a retroviral poly(A) trap (consisting of a RNA polymerase II promoter, a neomycin-resistance gene, and 5'-splice site) typically produced mutagenized clones in which vector sequences spliced to the 3'-terminal exons of cellular transcription units, suggesting strong selection for fusion transcripts that evade nonsense-mediated decay. The efficient exchange of poly(A) traps should greatly extend the utility of mutant libraries generated by gene entrapment and provides new strategies to study the rules that govern the expression of exons inserted throughout the genome.

Animals↗

PubMed2003

Computational discovery of internal micro-exons.

Very short exons, also known as micro-exons, occur in large numbers in some eukaryotic genomes. Existing annotation tools have a limited ability to recognize these short sequences, which range in length up to 25 bp. Here, we describe a computational method for the identification of micro-exons using near-perfect alignments between cDNA and genomic DNA sequences. Using this method, we detected 319 micro-exons in 4 complete genomes, of which 224 were previously unknown, human (170), the nematode Caenorhabditis elegans (4), the fruit fly Drosophila melanogaster (14), and the mustard plant Arabidopsis thaliana (36). Comparison of our computational method with popular cDNA alignment programs shows that the new algorithm is both efficient and accurate. The algorithm also aids in the discovery of micro-exon-skipping events and cross-species micro-exon conservation.

Alternative Splicing↗

PubMed1995

Expression of CD44 variant exons 8-10 in colorectal cancer and its relationship to metastasis.

Splice variants of CD44 are overexpressed in human lung, breast, and colon carcinoma cell lines. This study was conducted to clarify the association between the expression of CD44 variant exons 8-10 and metastatic potential in human colorectal cancer. We found that the expression of a CD44 splice variant containing exons v8-10 was increased in all of 60 colorectal cancer specimens examined compared with matched normal colerectal mucosa, as determined by Northern blotting. Expression of CD44 variant exons 8-10 did not significantly correlate with histological type, depth of tumor invasion, lymphatic invasion, venous invasion, or lymph node metastasis. However, the level of CD44 variant exon 8-10 expression was significantly higher in carcinomas associated with liver metastasis than in those without liver metastasis. In addition, expression of CD44 variant exons 8-10 in the liver metastases was more intense than that in the primary colorectal cancers. These findings indicated that this domain of the CD44 glycoprotein encoded by exons v8-10 may play an important role in tumor hematogenous metastasis of human colorectal cancer.

Blotting, Northern↗

PubMed1999

Somatic mutations in RET exons 12 and 15 in sporadic medullary thyroid carcinomas: different spectrum of mutations in sporadic type from hereditary type.

Germline mutations in the RET proto-oncogene are responsible for multiple endocrine neoplasia type 2 (MEN 2A and 2B) and familial medullary thyroid carcinoma (FMTC). Point mutations or in-frame deletions of exons 10, 11, 13, 14 and 16 are associated with sporadic medullary thyroid carcinoma (MTC). To understand further the role of the RET gene in sporadic MTC, we examined mutations in exons 12 and 15 of RET in patients with sporadic MTC. DNAs were extracted from 39 formalin-fixed tumor tissues and corresponding normal thyroid tissues or peripheral blood leukocytes. DNA sequencing was used to identify mutations in exons 12 and 15 of RET. In this study, one novel somatic mutation was found in exon 12 and five novel mutations or deletions were found in exon 15. Of the patients with mutations, one had an in-frame 12-bp deletion (nt. 2625-2636), one had point mutations in both codons 884 and 908, and the remaining three had point mutations in codons 748, 876 and 901, respectively. Together with our previous identification of somatic mutations in exons 10, 11, 13, 14 and 16, somatic alterations were found in 10 out of 39 (25.6%) sporadic MTCs. There was no association of RET gene mutations with tumor recurrence or prognosis. These results suggest that mutations occur frequently in the RET coding region in addition to the previously reported mutation hot spots, and there is a different spectrum of mutations between sporadic and hereditary MTC.

Adult↗

PubMed2006

A rare RET gene exon 8 mutation is found in two Greek kindreds with familial medullary thyroid carcinoma: implications for screening.

OBJECTIVE: Familial medullary thyroid carcinoma (FMTC) is caused by germ-line mutations in the RET proto-oncogene. These mutations concern mainly cysteine residues in exons 10 and 11, whereas noncysteine mutations in exons 13-16 are rare. Mutations in other exons have been reported only in isolated families. In this study we have analysed the RET gene in two FMTC families negative for mutations in the above exons. DESIGN: We have analysed exons 7-19 and 21 in one index patient from each family using DNA sequencing. PATIENTS: Twenty-eight subjects from both families were clinically assessed and subsequently molecularly analysed for the presence of RET gene mutations. RESULTS: We have found the mutation c.1597G-->T (Gly533Cys) in two Greek families with FMTC. The mutation was detected in all seven MTC patients of both families as well as in 13 asymptomatic relatives in the heterozygote state, although one of the patients was also a homozygote due to consanguinity. The mutation shows a wide clinical heterogeneity, as there are carrier patients with age of diagnosis ranging from 23 to 88 years. CONCLUSIONS: It is likely that this mutation causes FMTC, as no other mutation was found in the RET gene, the mutation co-segregates with FMTC, and family members without the mutation are clinically unaffected. As the same point mutation was previously found in a large Brazilian family, it may be present in other populations as well. Therefore, exon 8 of RET should be screened in FMTC families with no identified common RET mutations.

Adult↗

PubMed1990

Nucleotide sequence and exon-intron organization of the human proacrosin gene.

Acrosin is a serine proteinase and located in a zymogen form, proacrosin, in the acrosome of the sperm. As deduced from the cDNA sequences for human and boar proacrosin, the enzyme is synthesized as a preproenzyme, preproacrosin, which contains a hydrophobic leader sequence. Using cDNA clones as probes, we have isolated the gene coding for human proacrosin from a human leucocyte genomic library and a human cosmid library, respectively. The gene contains four introns between 0.2 kb--4.5 kb in length. Similar to other serine proteinases, the coding sequence of the preproacrosin gene is spread over all the five exons of the gene and the three activesite residues His, Asp and Ser are encoded by three different exons. According to the exon-intron structure, preproacrosin is suggested to be closely related to the serine proteinase subfamily containing trypsin and kallikrein. However, the light chain of proacrosin seems to be similar to that of chymotrypsin. The coding of the serine active-site residue together with the proacrosin-specific proline-rich domain in one exon, namely exon E5, let us assume that the nucleotide sequence for the proline-rich domain was generated during evolution by intron-exon transfer from a foreign gene with subsequent intron excision. By primer extension analysis, the transcription initiation site of the preproacrosin mRNA could be assigned to the residue C at -74 nucleotides upstream from the translation initiation codon ATG. In contrast to most other eucaryotic genes, including the known testis-specific genes, typical TATA and CAAT box sequences in convential distances from the 5' end of the transcription start site could not be evaluated in the proacrosin gene.

Acrosin↗

PubMed1997

The mouse gene for hypoxia-inducible factor-1alpha--genomic organization, expression and characterization of an alternative first exon and 5' flanking sequence.

The ubiquitously expressed hypoxia-inducible factor-1 (HIF-1) is involved in expression of a large number of oxygen-regulated genes. HIF-1 is a heterodimer consisting of an alpha and a beta subunit, both belonging to the basic-helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator-Sim (PAS) family of transcription factors. Whereas HIF-1alpha is a novel member of this family, HIF-1beta is identical to the aryl hydrocarbon receptor nuclear translocator, previously recognized to be involved in xenobiotic metabolism. cDNA cloning revealed that mouse HIF-1alpha can be expressed as two mRNA isoforms containing alternative 5' untranslated regions and two different predicted translational start sites. We cloned and characterized 20.5 kb of the mouse HIF-1alpha gene (Hif1a) containing exon II-XV. The two alternative first exons, I.1 and I.2, are separated from exon II by approximately 24 kb and 17 kb, respectively. We also sequenced Hif1a exon I.1 and flanking regions, and mapped a single exon I.1 transcription initiation site. Reverse transcription PCR analysis of total RNA derived from normoxic and hypoxic mouse hepatoma and fibroblast cell lines suggested that the two alternative mRNA isoforms are constitutively coexpressed in these cells, and that two different promoters drive transcription of HIF-1alpha. A minimal exon I.1 promoter was identified which moderately activated heterologous gene expression, indicating that additional cis-elements are required for efficient HIF-1alpha transcription in vivo.

Animals↗

PubMed1991

Identification of the new isoforms of mouse myelin basic protein: the existence of exon 5a.

Myelin basic protein (MBP) is a major constituent in the myelin of the CNS. In mice, five forms of MBPs (14 kDa, two types of 17 kDa, 18.5 kDa, and 21.5 kDa) encoded by separate mRNAs have been identified based on cDNA cloning studies. These mRNAs are considered to be produced by alternative splicing from a single gene composed of seven exons. Here we report the existence of two novel MBP mRNAs encoding 19.7-kDa and 21-kDa MBPs identified by cDNA cloning using the polymerase chain reaction. Both of these MBPs contain a sequence of a previously unidentified exon of 66 nucleotides, which was mapped to be just 5' of exon 5 in the MBP gene. MBP mRNAs containing this novel exon (exon 5a) belong to a minor population in the whole brain and PNS and are somewhat enriched in the spinal cord. Exon 5a encodes a very hydrophobic segment rich in valine residues, which presumably forms a beta-pleated sheet.

Animals↗

PubMed1998

Isolation, cloning, and sequencing of porcine agouti exon 2 (PorAex2).

In order to isolate, clone, and sequence agouti exon 2 of the pig (Yorkshire), we used an interspecific hybridization strategy. Primers from the 5' and 3' borders of the known human agouti exon 2 sequence were used to amplify (PCR) pig agouti exon 2. Following Southern blotting using a human exon 2 internal primer to authenticate that our PCR amplified product was truly pig exon 2 (PorAex2), the fragment was cloned and sequenced. PorAex2 exhibits 79.1 and 75.7% DNA sequence and 85 and 74% deduced amino acid sequence homologies with human and mouse agouti exon 2 and agouti protein, respectively. With the isolation of PorAex2, we can now map, sequence, and clarify the modus operandi of the porcine agouti gene. The GenBank Accession number of PorAex 2 is AF018166.

Agouti Signaling Protein↗

PubMed2006

Characterization of human deoxyribonuclease I gene (DNASE1) promoters reveals the utilization of two transcription-starting exons and the involvement of Sp1 in its transcriptional regulation.

Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown to be altered by physiological and/or pathological processes. However, no information is available on the regulation of DNase I gene (DNASE1) expression in vivo or in vitro. We first mapped the transcription start sites of DNASE1 in human pancreas and in the DNase I-producing human pancreatic cancer cell line QGP-1, and revealed a novel site approximately 12 kb upstream of exon 1, which was previously believed to be the single transcription-starting exon. This initiation site marks an alternative starting exon, designated 1a. Exons 1 and 1a were used simultaneously as transcription-starting exons in pancreas and QGP-1 cells. Promoter assay, EMSA and chromatin immunoprecipitation analysis with QGP-1 cells showed the promoter region of exon 1a in which the Sp1 transcription factor is specifically involved in promoter activity. This is the first to be identified as a transcription factor responsible for gene expression of vertebrate DNase I genes. Furthermore, RT-PCR analysis indicated alternative splicing of human DNASE1 pre-mRNA in pancreas and QGP-1 cells. Only two transcripts among eight alternative splicing products identified can be translated to produce intact DNase I protein. These results suggest that human DNASE1 expression is regulated through the use of alternative promoter and alternative splicing.

Alternative Splicing↗

PubMed1990

How big is the universe of exons?

If genes have been assembled from exon subunits, the frequency with which exons are reused leads to an estimate of the size of the underlying exon universe. An exon database was constructed from available protein sequences, and homologous exons were identified on the basis of amino acid identity; statistically significant matches were determined by Monte Carlo methods. It is estimated that only 1000 to 7000 exons were needed to construct all proteins.

Amino Acid Sequence↗