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PubMed1986

Synthesis of growth hormone-prolactin chimeric proteins and processing mutants by the exchange and deletion of genomic exons.

To test the feasibility of synthesizing recombinant peptide hormones by exon deletion and exchange, we have constructed and expressed hybrid human growth hormone (hGH)-rat prolactin (rPrl) genes in which the third and fourth exons of the hGH gene are deleted and separately replaced by the corresponding exons of the rPrl gene. These exon deletion and exon exchange genes were inserted into an SV40 viral vector, packaged, and expressed following acute viral infection of monkey kidney cells. Expression of the hGH gene lacking the third exon (hGHd3) was not detectable at the level of protein production. However, replacement of the deleted third exon in the hGHd3 gene with exon 3 of the rPrl gene (hGHP3) resulted in the efficient synthesis of a secreted chimeric protein. When the fourth exon of the hGH gene was deleted (hGHd4), the encoded protein was found only in the cytosol despite signal sequence processing. Replacement of the deleted fourth exon in this hGHd4 gene with exon 4 of rPrl resulted in the synthesis and secretion of both a chimeric protein (hGHP4) as well as a larger protein corresponding in size to prehGHP4. These results suggest that exon exchange among distantly related genes in the GH family may be used to produce high levels of chimeric GH-related proteins, and regions internal to the hGH protein may be critical in establishing normal protein processing and secretion.

Chimera↗

PubMed1999

Mutation analysis of the BRCA1 and BRCA2 genes results in the identification of novel and recurrent mutations in 6/16 flemish families with breast and/or ovarian cancer but not in 12 sporadic patients with early-onset disease. Mutations in brief no. 224. Online.

Since the identification of the BRCA1 and BRCA2 genes (MIM#s 113705 and 600185), more than hundred different mutations throughout both genes have been reported. Recurrent mutations are rare and mainly due to founder effects. We analyzed 12 sporadic female patients with breast cancer before age 35, as well as 16 unrelated families, presenting with either (i) at least 3 first degree relatives with breast and/or ovarian cancer diagnosed at any age, or (ii) at least 2 first and/or second degree relatives with breast and/or ovarian cancer before age 45 years. We performed a protein truncation test for BRCA1 exon 11 and BRCA2 exons 10 and 11 and heteroduplex analysis for all the remaining exons of BRCA1 and 2. Presence of genomic deletions encompassing exons 13 or 22 of BRCA1, known to be Dutch founder mutations, was investigated by PCR. In 6/16 (37.5%) unrelated families the causal mutation in either the BRCA1 or BRCA2 gene was identified. Four different mutations were found in the BRCA1 gene: IVS5+3A>G (intron 5), 1191delC (exon 11), R1443X (exon 13), IVS22+5G>A (intron 22) and two in the BRCA2 gene: 6503delTT (exon 11), 6831delTG (exon 11). 1191delC (BRCA1) and 6831delTG (BRCA2) are novel mutations. IVS5+3A>G in exon 5 of BRCA1 published by Peelen et al. (1997) as a novel Belgian mutation, was identified in one additional family, not fulfilling our inclusion criteria. In the group of 12 sporadic female patients no mutations were found.

Adult↗

PubMed1998

Spinal muscular atrophy due to an isolated deletion of exon 8 of the telomeric survival motor neuron gene.

Patients with autosomal recessive spinal muscular atrophy (SMA) usually carry a homozygous deletion of exons 7 and 8 of the telomeric survival motor neuron (SMN(T)) gene, although an isolated deletion of SMN(T) exon 8 has never been found. We now report on 2 patients with the typical features of SMA types II and III, who carried a homozygous deletion of SMN(T) exon 8 but retained SMN(T) exon 7. Importantly, to exclude a sequence conversion event of telomeric exon 8, we amplified a fragment that spanned exons 7 and 8 of the SMN gene. The resulting 1,010-base pair (bp) fragments were subjected to nested polymerase chain reaction (PCR) of exon 7. The subsequent restriction analysis failed to show any products of telomeric exon 7, as the site for primer 541C1120 was lost in both alleles. These findings indicate a homozygous deletion of SMN(T) exon 8. Direct sequencing of the cloned 1,010-bp fragment further confirmed that these 2 SMA patients did not possess telomeric exon 8. The more severely affected child also showed a deletion of the neuronal apoptosis inhibitory protein (NAIP) gene. The present findings provide evidence that an isolated deletion of SMN(T) exon 8 is associated with the milder subtypes of SMA. Our data also demonstrate that the additional deletion of the NAIP gene exacerbates the severity of the disease.

Adult↗

PubMed1994

Genomic organization of IFI16, an interferon-inducible gene whose expression is associated with human myeloid cell differentiation: correlation of predicted protein domains with exon organization.

The human IFI16 gene is a member of an interferon-inducible family of mouse and human genes closely linked on syntenic regions of chromosome 1. Expression of these genes is largely restricted to hemopoietic cells, and is associated with the differentiation of cells of the myeloid lineages. As a prelude to defining the mechanisms governing IFI16 expression, we have deduced its genomic organization using a combination of genomic cloning and polymerase chain reaction amplification of genomic DNA. IFI16 consists of ten exons and nine intervening introns spanning at least 28 kilobases (kb) of DNA. The reiterated domain structure of IFI16 protein is closely reflected in its intron/exon boundaries, and may represent the evolutionary fusion of several independent functional domains. Thus, exon 1 consists of 5' untranslated (UT) sequences and contains sequence motifs that may confer interferon-inducibility, and exon 2 encodes the lysine-rich amino-terminal ("K") region, which possesses DNA-binding activity. Exon 3 codes for a domain which is poorly conserved between family members, except for a strongly retained basic motif likely to provide localization. The first of two 200 amino acid repeat domains that are the hallmark of this family (domain A) is represented jointly on exons 4 and 5, which are reiterated as exons 8 and 9, respectively, to encode the second 200 amino acid domain (B). Two intervening serine-threonine-rich domains (C and C'), unique to IFI16, are each encoded by single exons of identical length (exons 5 and 6). These domains are predicted to encode semi-rigid "spacer" domains between the 200 amino acid repeats. The reiterated nature of exons 4 to 6 and the insertion of introns into a single reading frame strongly suggest that IFI16 and related genes arose by a series of exon duplications, some of which antedated speciation into mouse and humans. Several alternative mRNA cap sites downstream of a TATA consensus sequence were defined, using primer extension analysis of mRNA. Sequencing of approximately 1.7 kb of DNA upstream of this region revealed no recognizable consensus elements for induction by interferon-alpha (interferon-alpha/beta-stimulated response elements), but two motifs resembling interferon-gamma activation sites were located. IFNs alpha and gamma both induce IFI16 mRNA expression in myeloid cells. Interferon-alpha inducibility of IFI16 may be regulated by an interferon-alpha/beta-stimulated response consensus element in the 5' UT exon, as a similar motif is conserved in the corresponding position in the related myeloid cell nuclear differentiation antigen gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1999

An exon map of the AZFc male infertility region of the human Y chromosome.

STS markers spanning the AZFc region of human Y Chromosome (Chr) were used to isolate a series of 14 P1 artificial chromosome (PAC) clones covering at least 560 kb of DNA. End clone analysis of these PAC clones was used to derive 10 new STS markers. Together with other markers mapped to the region, an STS content analysis was used to assemble these PAC clones into three distinct contigs. A minimum tiling path of ten PAC clones was subjected to exon trapping to identify potentially new genes mapping to the AZFs region. In all, 39 potential exons were isolated, including 2 exons from the DAZ gene, 3 exons from the BPY2 gene, 2 exons from the PRY gene, and 1 exon from a member of the RBM II gene family; all these genes have been shown previously to map to the AZFc region. One further exon was found that shows homology to the DFFRY gene, which maps to Yq11. 2, indicating that there may be a further copy of this gene or a pseudogene in the distal Yq euchromatin. The majority of the remaining potential exons appear to be novel, suggesting that additional genes lie in the AZFc region. Mapping of these exons by PCR analysis of somatic cell hybrids has shown that six of these exons are homologous to autosomal sequences, and five to sequences on the X Chr. RT-PCR analysis of primary cDNA from adult testis, brain, liver, and skeletal muscle mRNA has shown that 11 of the novel exons are expressed in one or a combination of these tissues, indicating that they form parts of genuine transcripts.

Brain↗

PubMed2002

FOXP2: novel exons, splice variants, and CAG repeat length stability.

FOXP2 is a transcription factor containing a polyglutamine tract, a zinc-finger motif, and a forkhead DNA-binding domain. The FOXP2 gene is located on 7q31. A missense mutation in the forkhead domain (exon 14) and a balanced reciprocal translocation t(5;7)(q22;q31.2) with a breakpoint between exons 3b and 4 have recently been associated with a speech and language disorder (SPCH1). The role of FOXP2 in this neurodevelopmental disorder suggests that mutations in FOXP2 could cause other neuropsychiatric disorders. To begin investigation of this possibility, we examined the genomic structure and CAG/CAA repeat region of FOXP2. We detected little polymorphism and no expansions in the FOXP2 CAG/CAA repeat in 142 individuals with progressive movement disorders. We found evidence of alternate splice variants and six previously undetected exons: three 5' untranslated exons (s1, s2, s3), two additional untranslated exons (2a and 2b) between exons 2 and 3, a translated exon (4a) between exons 4 and 5, and a longer version of exon 10 (10+) that contains an alternate stop codon and produces a truncated protein (FOXP2-S). Our results suggest that FOXP2 spans at least 603 kb of genomic DNA, more than twice the previously defined region, and provide evidence of a promoter region flanking exon s1. This demonstration of additional FOXP2 exons and splice variants should facilitate understanding of FOXP2 function and the search for additional FOXP2 mutations.

Alternative Splicing↗

PubMed2004

Conservation and expression of an alternative 3' exon of Runx2 encoding a novel proline-rich C-terminal domain.

The Runx2 (Cbfa1, Aml3, PEBP2alphaA) gene plays an essential role in bone development and is one of a three-member family of closely related genes that encode the alpha-chain DNA binding components of the heterodimeric core binding factor complex. While all three mammalian Runx genes share a complex dual promoter structure (P1, P2) and display alternative splicing, a distinctive feature of Runx2 is the potential to encode larger isoforms in which the C-terminal domain encoded by the standard 3' terminal exon (exon 6) is replaced by an extended 200-201 amino acid C-terminal sequence including an extensive proline-rich domain and a C-terminal amphipathic helix. We report that the novel exon that gives rise to these variants (exon 6.1) is located over 100 kb downstream of exon 6 in the mouse, rat and human genomes. Exon 6.1 spans a CpG-rich island, and human/rodent conservation is evident through the coding sequence and the 3' untranslated region (UTR). Reverse transcriptase polymerase chain reaction (RT-PCR) and blot hybridisation analyses reveal that exon 6.1 is utilised at low levels in all mouse tissues and cell lines that express Runx2, regardless of which promoter is active, giving Runx2 the potential to encode more than 12 distinct isoforms. RT-PCR analysis of human RUNX2 exon 6.1 expression shows that utilisation of this exon is also conserved. In vitro transcription/translation of cDNAs encoding several exon 6.1 isoforms reveals that the novel Runx proteins are able to bind specifically to canonical Runx DNA target sequences. Antibodies raised to the unique C-terminal domain were shown to be reactive by immunoprecipitation and immunoblot assay, and were used in confocal immunofluorescence microscopy to reveal low level cytoplasmic staining in osteosarcoma and lymphoma cells that express high levels of Runx2 mRNA. However, reactive protein could not be detected in immunoblots of extracts from either cell type, suggesting that these proteins are unstable in lymphoid and osteosarcoma cells. In conclusion, the conservation and widespread utilisation of Runx2 exon 6.1 suggest that its encoded isoforms play an as yet undetermined role in mammalian development.

Alternative Splicing↗

PubMed1999

Combinatorial control of a neuron-specific exon.

The mouse c-src gene contains a short neuron-specific exon, N1. N1 exon splicing is partly controlled by an intronic splicing enhancer sequence that activates splicing of a heterologous reporter exon in both neural and nonneural cells. Here we attempt to dissect all of the regulatory elements controlling the N1 exon and examine how these multiple elements work in combination. We show that the 3' splice site sequence upstream of exon N1 represses the activation of splicing by the downstream intronic enhancer. This repression is stronger in nonneural cells and these two regulatory sequences combine to make a reporter exon highly cell-type specific. Substitution of the 3' splice site of this test exon with sites from other exons indicates that activation by the enhancer is very dependent on the nature of the upstream 3' splice site. In addition, we identify a previously uncharacterized purine-rich sequence within exon N1 that cooperates with the downstream intronic enhancer to increase exon inclusion. Finally, different regulatory elements were tested in multiple cell lines of both neuronal and nonneuronal origin. The individual splicing regulatory sequences from the src gene vary widely in their activity between different cell lines. These results demonstrate how a simple cassette exon is controlled by a variety of regulatory elements that only in combination will produce the correct tissue specificity of splicing.

Animals↗

PubMed1999

Trans-complementation of the second step of pre-mRNA splicing by exogenous 5' exons.

During splicing of nuclear pre-mRNAs, the first step liberates the 5' exon (exon 1) and yields a lariat intron-3'exon (intron-exon 2) intermediate. The second step results in exon ligation. Previous results indicated that severe truncations of the 5' exon of the actin pre-mRNA result in a block to the second splicing step in vitro in yeast extracts, leading to an accumulation of intron-exon 2 lariat intermediates. We show that exogenous exon 1 RNA oligonucleotides can chase these stalled intermediates into lariat intron and spliced exons. This reaction requires some of the cis elements and trans-acting factors that are required for a normal second step. There is no strong sequence requirement for the exon 1 added in trans, but oligonucleotides with complementarity to the U5 snRNA conserved loop perform the chase more efficiently. Using a dominant negative mutant of the DEAH-box ATPase Prp16p and ATP depletion, we show that the stalled intermediate is blocked after the Prp16p-dependent step. These results show that exogenous RNAs with various sequences but containing no splicing signals can be incorporated into spliceosomes and undergo RNA recombination and exon shuffling during the second step of pre-mRNA splicing.

Base Sequence↗

PubMed2006

Gastrointestinal stromal tumors with KIT exon 11 deletions are associated with poor prognosis.

BACKGROUND & AIMS: Gain-of-function mutations in the KIT receptor tyrosine kinase gene and rare mutations in the platelet-derived growth factor receptor alpha (PDGFRA) gene are important events in gastrointestinal stromal tumor (GIST) development. Different mutations are reportedly associated with distinctive phenotypes and possibly clinical behavior. We investigated the correlation among mutation type, phenotype, and clinical course in a preimatinib, population-based series of GIST with long-term follow-up. METHODS: Genomic DNA from 177 GIST patients was analyzed for KIT exons 9, 11, 13, and 17 and PDGFRA exons 12 and 18 mutations using denaturating high-performance liquid chromatography and bidirectional sequencing. RESULTS: KIT exon 11 mutations were detected in 101 of 177 GIST (61 deletions, 23 missense mutations, and 17 duplications); wild-type (WT) KIT and PDGFRA were detected in 63; KIT exon 9 and exon 17 mutations in 6 and 1, respectively; and PDGFRA exons 12 and 18 mutations in 3 each. GIST >5 cm vs GIST </=1 cm had mutations in 73% and 33%, respectively. KIT exon 11 deletions were significantly associated with a higher proportion of high risk or overtly malignant groups compared with WT GIST. KIT exon 11 deletions adversely affected outcome. KIT exon 11 duplications and exon 9 mutations were found exclusively in gastric and small intestinal GIST, respectively. CONCLUSIONS: KIT exon 11 deletion is an independent adverse prognostic factor in patients with GIST.

Adult↗

PubMed2006

Comparison of multiple vertebrate genomes reveals the birth and evolution of human exons.

Orthologous gene structures in eight vertebrate species were compared on a genomic scale to detect the birth and maturation of new internal exons during the course of evolution. We found that 40% of new human exons are alternatively spliced, and most of these are cassette exons (exons that are either included or skipped in their entirety) with low inclusion rates. This proportion decreases steadily as older and older exons are examined, even as splicing efficiency increases. Remarkably, the great majority of new cassette exons are composed of highly repeated sequences, especially Alu. Many new cassette exons are 5' untranslated exons; the proportion that code for protein increases steadily with age. New protein-coding exons evolve at a high rate, as evidenced by the initially high substitution rates (K(s) and K(a)), as well as the SNP density compared with older exons. This dynamic picture suggests that de novo recruitment rather than shuffling is the major route by which exons are added to genes, and that species-specific repeats could play a significant role in recent evolution.

Alternative Splicing↗

PubMed1988

Exon-intron organization and sequence comparison of human and murine T11 (CD2) genes.

Genomic DNA clones containing the human and murine genes coding for the 50-kDa T11 (CD2) T-cell surface glycoprotein were characterized. The human T11 gene is approximately equal to 12 kilobases long and comprised of five exons. A leader exon (L) contains the 5'-untranslated region and most of the nucleotides defining the signal peptide [amino acids (aa) -24 to -5]. Two exons encode the extracellular segment; exon Ex1 is 321 base pairs (bp) long and codes for four residues of the leader peptide and aa 1-103 of the mature protein, and exon Ex2 is 231 bp long and encodes aa 104-180. Exon TM is 123 bp long and codes for the single transmembrane region of the molecule (aa 181-221). Exon C is a large 765-bp exon encoding virtually the entire cytoplasmic domain (aa 222-327) and the 3'-untranslated region. The murine T11 gene has a similar organization with exon-intron boundaries essentially identical to the human gene. Substantial conservation of nucleotide sequences between species in both 5'- and 3'-gene flanking regions equivalent to that among homologous exons suggests that murine and human genes may be regulated in a similar fashion. The probable relationship of the individual T11 exons to functional and structural protein domains is discussed.

Amino Acid Sequence↗

PubMed1991

Relationship between alternatively spliced exons and functional domains in tropomyosin.

Smooth and striated muscle alpha-tropomyosins differ as a consequence of alternative splicing of exons 2 and 9 encoding amino acid residues 39-80 and 258-284, respectively [Ruiz-Opazo, N., Weinberger, J. & Nadal-Ginard, B. (1985) Nature (London) 315, 67-70]. To understand the relationship between alternatively spliced exons and functional domains in tropomyosin, recombinant unacetylated striated muscle, smooth muscle, and chimeric rat alpha-tropomyosins (+H3N-tropomyosins) expressed in and purified from Escherichia coli were analyzed. The functional differences between the isoforms can be primarily ascribed to exon 9. +H3N-Tropomyosins with the smooth muscle exon 9 bound to skeletal muscle filamentous actin with at least a 5-fold higher affinity than +H3N-tropomyosins with the striated muscle exon 9. On the other hand, in the presence of Ca2+, troponin increased the affinity of +H3N-tropomyosins with the striated muscle exon 9 at least 50-fold, whereas it had little effect on +H3N-tropomyosins with the smooth muscle exon 9. The unique striated muscle alpha-tropomyosin exon 9 seems to be specialized for Ca(2+)-insensitive interaction with troponin on the thin filament. The unique smooth muscle alpha-tropomyosin exon 2 was associated with a slightly lower actin affinity than the striated muscle exon 2. Although the regions encoded by exons 2 and 9 correspond to functional domains, they are not recognizable as independent units or structural domains in the extended coiled-coil structure of this fibrous actin binding protein.

Actins↗

PubMed1999

Redundant intronic repressors function to inhibit fibroblast growth factor receptor-1 alpha-exon recognition in glioblastoma cells.

The human fibroblast growth factor receptor-1 primary transcript is alternatively processed to produce receptor forms that vary in their affinity for fibroblast growth factor. The inclusion of a single exon (alpha) in normal brain glial cells produces a low affinity form of the receptor. Recognition of the alpha-exon is dysregulated during neoplastic transformation of glial cells to produce a high affinity receptor form. In this study, we have identified a second intronic repressor of RNA splicing located approximately 250 nucleotides upstream of the alpha-exon. Deletion or mutation of this sequence resulted in a significant increase in exon recognition in glioblastoma cells. This intronic repressor was found to share significant sequence homology with an intronic repressor element located downstream of the alpha-exon. The two repressor elements are functionally redundant in that they are capable of inhibiting alpha-exon recognition when positioned upstream or downstream of the exon. Finally, the elements were found to mediate enhanced exclusion of an unrelated exon, but only the repressors were placed flanking the exon. However, under these conditions, the cell-specific exon exclusion was no longer maintained. These results suggest that although the alpha-exon inclusion is actively repressed in glioblastomas, the absence of trans-activators appears to be key to the production of the high affinity form of fibroblast growth factor receptor-1 in glioblastomas.

Alternative Splicing↗

PubMed2002

Complex regulation of human neuronal nitric-oxide synthase exon 1c gene transcription. Essential role of Sp and ZNF family members of transcription factors.

Neuronal nitric-oxide synthase (nNOS) is expressed in a variety of human tissues and shows a complex transcriptional regulation with the presence of nine alternative first exons (1a-1i) resulting in nNOS transcripts with differing 5'-untranslated regions. We previously demonstrated that nNOS exon 1c, one of the predominant transcripts in the human gastrointestinal tract, is driven by a separate promoter (Saur, D., Paehge, H., Schusdziarra, V., and Allescher, H. D. (2000) Gastroenterology 118, 849-858). The present study focused on the quantitative expression of nNOS first exon variants in different human tissues and the characterization of the basal nNOS exon 1c promoter. In human brain, skeletal muscle, colon, and TGW-nu-I neuroblastoma cells, first exon expression patterns were analyzed by quantitative real-time reverse transcription-PCR. In these tissues/cells exon 1c was one of the most abundant first exons of nNOS. By transient transfections of TGW-nu-I and HeLa cells with reporter plasmids containing a series of 5' and 3' deletions in the exon 1c regulatory region, the minimal TATA-less promoter was localized within 44 base pairs. Gel mobility shift assays of this cis-regulatory region revealed a high complexity of the basal promoter with a cooperative binding of several transcription factors, like Sp and ZNF family members. When the Sp binding site of the minimal promoter construct was mutated, promoter activity was completely abolished in both cell lines, whereas mutation of the common binding site of ZNF76 and ZNF143 resulted in a decrease of 53% in TGW-nu-I and 37% in HeLa cells. In Drosophila Schneider cells expression of Sp1, the long Sp3 isoform, ZNF76 and ZNF143 potently transactivated the nNOS exon 1c promoter. These results identify the critical regulatory region for the nNOS exon 1c basal promoter and stress the functional importance of multiple protein complexes involving Sp and ZNF families of transcription factors in regulating nNOS exon 1c transcription.

Base Sequence↗

PubMed2006

RBM4 interacts with an intronic element and stimulates tau exon 10 inclusion.

Tau protein, which binds to and stabilizes microtubules, is critical for neuronal survival and function. In the human brain, tau pre-mRNA splicing is regulated to maintain a delicate balance of exon 10-containing and exon 10-skipping isoforms. Splicing mutations affecting tau exon 10 alternative splicing lead to tauopathies, a group of neurodegenerative disorders including dementia. Molecular mechanisms regulating tau alternative splicing remain to be elucidated. In this study, we have developed an expression cloning strategy to identify splicing factors that stimulate tau exon 10 inclusion. Using this expression cloning approach, we have identified a previously unknown tau exon 10 splicing regulator, RBM4 (RNA binding motif protein 4). In cells transfected with a tau minigene, RBM4 overexpression leads to an increased inclusion of exon 10, whereas RBM4 down-regulation decreases exon 10 inclusion. The activity of RBM4 in stimulating tau exon 10 inclusion is abolished by mutations in its RNA-binding domain. A putative intronic splicing enhancer located in intron 10 of the tau gene is required for the splicing stimulatory activity of RBM4. Immunohistological analyses reveal that RBM4 is expressed in the human brain regions affected in tauopathy, including the hippocampus and frontal cortex. Our study demonstrates that RBM4 is involved in tau exon 10 alternative splicing. Our work also suggests that down-regulating tau exon 10 splicing activators, such as RBM4, may be of therapeutic potential in tauopathies involving excessive tau exon 10 inclusion.

Adult↗

PubMed2002

Pig amelogenin gene expresses a unique exon 4.

The pig amelogenin gene was isolated from a Lambda genomic library, and a 6.3 kb SalI/XbaI restriction fragment, inclusive of exons 3 through 7, was subcloned into a plasmid vector. DNA sequencing revealed two putative exon 4 sequences. The derived amino acid sequence of exon 4a, KSGRWGARLTAFVSSVQ, had previously been identified in a 190-amino-acid amelogenin isoform by protein sequencing. Exon 4b encoded the peptide DLYLEAIRIDRTAF, which is homologous to exon 4-encoded segments reported for human, mouse, and rat. Oligonucleotides from both of these exons were used to amplify cDNA generated from developing teeth. Amplification products were analyzed by agarose gel electrophoresis, cloned, and characterized by DNA sequencing. Exon 4a was found in transcripts encoding amelogenin isoforms having 190 and 73 amino acids. Exon 4b was found only in apparent splicing intermediates that retained intron 3, but was not detected in any final mRNA transcripts. Pig amelogenin having apparent molecular mass of 23 kD were isolated from the enamel matrix and characterized by mass spectrometry. Two mass values, 18,512.5, and 18,571.2 Da, were measured that match the values predicted for the 162-amino-acid cleavage product of the 173-amino-acid amelogenin, and the 165-amino-acid cleavage product of the 190-amino-acid amelogenin, which includes 17 amino acids encoded by exon 4a. We conclude that the pig amelogenin gene expresses a unique exon 4 that is not homologous to, or evolved from, the exon 4 segment expressed in humans and rodents.

Alternative Splicing↗

PubMed2005

Determinants of exon 7 splicing in the spinal muscular atrophy genes, SMN1 and SMN2.

Spinal muscular atrophy is a neurodegenerative disorder caused by the deletion or mutation of the survival-of-motor-neuron gene, SMN1. An SMN1 paralog, SMN2, differs by a C-->T transition in exon 7 that causes substantial skipping of this exon, such that SMN2 expresses only low levels of functional protein. A better understanding of SMN splicing mechanisms should facilitate the development of drugs that increase survival motor neuron (SMN) protein levels by improving SMN2 exon 7 inclusion. In addition, exonic mutations that cause defective splicing give rise to many genetic diseases, and the SMN1/2 system is a useful paradigm for understanding exon-identity determinants and alternative-splicing mechanisms. Skipping of SMN2 exon 7 was previously attributed either to the loss of an SF2/ASF-dependent exonic splicing enhancer or to the creation of an hnRNP A/B-dependent exonic splicing silencer, as a result of the C-->T transition. We report the extensive testing of the enhancer-loss and silencer-gain models by mutagenesis, RNA interference, overexpression, RNA splicing, and RNA-protein interaction experiments. Our results support the enhancer-loss model but also demonstrate that hnRNP A/B proteins antagonize SF2/ASF-dependent ESE activity and promote exon 7 skipping by a mechanism that is independent of the C-->T transition and is, therefore, common to both SMN1 and SMN2. Our findings explain the basis of defective SMN2 splicing, illustrate the fine balance between positive and negative determinants of exon identity and alternative splicing, and underscore the importance of antagonistic splicing factors and exonic elements in a disease context.

Base Sequence↗