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PubMed2006

Mutation in the U2 snRNA influences exon interactions of U5 snRNA loop 1 during pre-mRNA splicing.

The U2 and U6 snRNAs contribute to the catalysis of intron removal while U5 snRNA loop 1 holds the exons for ligation during pre-mRNA splicing. It is unclear how different exons are positioned precisely with U5 loop 1. Here, we investigate the role of U2 and U6 in positioning the exons with U5 loop 1. Reconstitution in vitro of spliceosomes with mutations in U2 allows U5-pre-mRNA interactions before the first step of splicing. However, insertion in U2 helix Ia disrupts U5-exon interactions with the intron lariat-3' exon splicing intermediate. Conversely, U6 helix Ia insertions prevent U5-pre-mRNA interactions before the first step of splicing. In vivo, synthetic lethal interactions have been identified between U2 insertion and U5 loop 1 insertion mutants. Additionally, analysis of U2 insertion mutants in vivo reveals that they influence the efficiency, but not the accuracy of splicing. Our data suggest that U2 aligns the exons with U5 loop 1 for ligation during the second step of pre-mRNA splicing.

Base Sequence↗

PubMed2007

The influence of antisense oligonucleotide length on dystrophin exon skipping.

Antisense oligonucleotides (AOs) can be used to redirect dystrophin pre-messenger RNA (mRNA) processing, to remove selected exons from the mature dystrophin mRNA, to overcome nonsense mutations, and/or restore the reading frame. Redundancy within the dystrophin protein allows some domains to be removed without seriously compromising function. One of the challenges for splicing blockade is to design AOs that efficiently remove targeted exons across the dystrophin pre-mRNA. AOs are initially designed to anneal to the more obvious motifs implicated in the splicing process, such as acceptor or donor splice sites and in silico predicted exonic splicing enhancers. The AOs are evaluated for their ability to induce targeted exon skipping after transfection into cultured myoblasts. Although no single motif has been implicated in the consistent induction of exon skipping, the length of the AO has emerged as an important parameter in designing compounds that redirect dystrophin pre-mRNA processing. We present data from in vitro studies in murine and human cells showing that appropriately designed AOs of 25-31 nucleotides are generally more effective at inducing exon skipping than shorter counterparts. However, there appears to be an upper limit in optimal length, which may have to be established on a case-by-case basis.

Animals↗

PubMed1997

A nonsense mutation in the 3-hydroxy-3-methylglutaryl-CoA lyase gene produces exon skipping in two patients of different origin with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.

A novel nonsense mutation associated with the skipping of constitutive exon 2 of the 3-hydroxy-3-methylglutaryl-CoA lyase gene was found in two patients, from Portugal and Morocco, with 3-hydroxy-3-methylglutaric acidemia. By reverse transcriptase PCR and single-strand conformational polymorphism a G-T transversion was located, at nucleotide 109, of the 3-hydroxy-3-methylglutaryl-CoA lyase cDNA, within exon 2. Two mRNAs were produced as a result of this nonsense mutation: one of the expected size that contains the premature stop codon UAA, and the other with a deletion of 84 bp corresponding to the whole of exon 2. This deletion produced the loss of the last seven amino acids of the leader peptide and the first 21 amino acids of the mature protein. The nonsense mutation was found in a purine-rich GGAAG sequence, which is equal to, or similar to, others reported to be exonic splicing enhancers (ESE). We suggest that the nonsense mutation may affect a possible ESE on exon 2, which would hinder the splice site selection and facilitate an aberrant splice with the skipping of this exon. Determination by quantitative PCR shows that the ratio of mRNA with the nonsense mutation to the mRNA with the deletion is approx. 3:1.

Amino Acid Sequence↗

PubMed2000

Analysis of the genetic variability of the 1st (CCC/ACC, P52T) and the 10th exons (bp 1012-1704) of the TSH receptor gene in Graves' disease.

We determined the genetic variability of the 1st (CCC/ACC, P52T polymorphic variant) and 10th exons (bp 1012-1704) of the TSH receptor (TSHR) gene in Graves' disease. A total of 101 Graves' patients and 163 control subjects were screened. The A253 mutant allele was carried by nine patients with Graves' disease (8.91%) and 13 control subjects (7.98%) in heterozygous genotype. No significant difference in the frequency of the mutant allele was found between Graves' patients and control subjects. These results provide evidence that the A253 polymorphism has no genetic relevance in Graves' disease. Moreover, the DNA nucleotide sequence of 693 bp of the 10th exon (bp 1012-1704) of the TSHR gene was determined in 15 Graves' patients. Six patients were homozygous for the wild-type allele and nine were heterozygous for the mutant allele at the 253rd nucleotide of the first exon. No polymorphism was found in the DNA sequences obtained from leukocytes of Graves' patients, similarly to the sequences obtained from the nine control subjects. None of the nine patients carrying the A253 polymorphism in the 1st exon of the TSHR had polymorphism in the examined part of the 10th exon, including two additional patients whose thyroid tissue was directly analysed. In all likelihood, the polymorphisms of the examined regions of either the 1st or the 10th exon of the THSR gene do not contribute to the genetic susceptibility to Graves' disease.

Adolescent↗

PubMed1996

Transient expression of CD44 variant isoforms in the ontogeny of the rat: ectoderm-, endoderm- and mesoderm-derived cells express different exon combinations.

Expression on rat tumor cells of CD44 variant isoforms containing exons v4-v7 or v6-v7 has been described as sufficient for initiation of the metastatic cascade. The question arose as to whether physiological programs may be reactivated by particular CD44 isoforms. With this in mind, expression of mRNAs for the CD44 isoforms was surveyed during ontogeny of the rat. Using available monoclonal antibodies, expression of the CD44 standard isoform (CD44s) and of an epitope of CD44 exon v6 (CD44v6) were evaluated by immunohistology also. While CD44s was expressed in cells of all three germ layers, CD44v6 expression was restricted to distinct epithelial layers and cells of the hematopoietic system. During ontogeny, expression of CD44v6 was first noted in the neural tube and the leading epithelial layer of the limb buds. Later, anti-CD44v6 (1.1ASML) stained basal layers of the epidermis, the epithelium of the gut, and the acini of the submandibular gland. Strong, but transient expression of CD44v6 was seen during lung development, in hematopoietic stem cells of the liver, in thymic epithelia and early thymic immigrants. Expression in these organs was downregulated shortly before or after birth. As revealed by Southern blotting after use of the reverse transcriptase polymerase chain reaction (RT-PCR), most CD44v6-positive organs contained more than one CD44 variant isoform, and the expression patterns differed between individual organs. Hematopoietic cells preferentially expressed exons v4-v7, endodermal tissue exons v4-v10 and only in the epidermis were exons v1-v10 detected. The temporally regulated expression during ontogeny and the different exon compositions suggest a pivotal role of CD44 isoforms particularly in hematopoesis and in pattern formation by instructive epithelia.

Animals↗

PubMed1996

Promoter region and alternatively spliced exons of the rat mu-opioid receptor gene.

The actions of exogenous and endogenous opioids are mediated by at least three different opioid receptors, called mu, kappa, and delta. Recently, we have detected a new variant of the rat mu-opioid receptor, which we termed rMOR1B and which differs from rMOR1 (now also called rMOR1A) in the amino acid sequence at the C-terminus. Both isoforms were proposed to be splicing variants of the same gene. To elucidate the molecular mechanism leading to the formation of the new variant, the exon/intron structure of the rat mu-opioid receptor gene in the respective area has been determined by analyzing a genomic P1 phage clone. In addition, we have investigated the putative promoter region of this gene. The present study revealed that rMOR1B is generated by an alternative splicing event whereby a previously unknown exon will be placed behind exon 3 to form rMOR1B mRNA, which is separated from the latter by an intron. Therefore, this new exon has to be called exon 4, whereas the former exon 4, which encodes the C-terminus of MOR1A, now becomes exon 5. Examination of the putative rat promoter region revealed a high degree of nucleotide sequence homology to the mouse gene. Using an RNase protection approach, one single transcription initiation site could be located at 230 bp upstream of the translation start. This is similar to the situation in the mouse, where four major transcription start sites were reported to lie close together around 270 bp upstream of the protein coding region.

Alternative Splicing↗

PubMed2002

Analysis on the promoter region of exon IV brain-derived neurotrophic factor in NG108-15 cells.

We have reported that the nuclear isoforms of Ca2+/calmodulin-dependent protein kinase II (CaM KII) are involved in the expression of the exon IV-containing brain-derived neurotrophic factor (BDNF) mRNA. We document here the cis-elements and transcription factors responsive to CaM KII in the activation of the promoter upstream of the exon IV (exon IV-BDNF promoter). Effects of constitutively active mutants of CaM KIV, MAPK kinase kinase (MEKK) and protein kinase A (PKA) on the exon IV-BDNF promoter activity were also evaluated by transfection and luciferase assay. The exon IV-BDNF promoter activity was increased by transfection with CaM KII, MEKK and PKA, but not by CaM KIV. Deletion and mutational analysis of the promoter revealed that the region between nucleotides -56 and -27 was responsive to CaM KII, which contained a CCAAT-box in the region between nucleotides -56 and -43. Expression of C/EBPbeta increased the promoter activity and potentiated the effects of CaM KII. The region between nucleotides -79 and -56 was responsive to MEKK, in which both a GA-rich sequence and a GC-box were included. Expression of Sp1 increased the promoter activity, which was further enhanced by transfection with MEKK. The region between nucleotides -43 and -27 was responsive to both PKA and CaM KII, but the transcription factors involved in the region remained unclear. These results suggest that the promoter of the exon IV-BDNF is at least regulated by CaM KII, MEKK and PKA, and that C/EBP/beta and Sp1 are potential transcription factors activated by CaM KII and MEKK, respectively.

Animals↗

PubMed1997

Co-presence of a point mutation and a deletion of exon 3 in the glycophorin C gene and concomitant production of a Gerbich-related antibody.

BACKGROUND: Antigens of the human blood group system Gerbich (Ge) are located on sialoglycoproteins glycophorin C (GPC) and glycophorin D (GPD). CASE REPORT: The Ge+ proposita (RW) produced an alloanti-Ge after receiving 2 units of red cells (RBCs) during surgery. Further studies were carried out to characterize the antibody specificity, RBC GPC and/or GPD (GPC/GPD), and the glycophorin C gene (GYPC) from RW and her compatible siblings. RW's serum contained an alloanti-Ge that did not react with RBCs from RW or four of her siblings or with RBCs with Ge-negative phenotypes. An eluate of RW's antibody reacted weakly with GPC in Western blotting. RW's RBCs were positive with 20 alloanti-Ge2, 1 autoanti-Ge2, 4 alloanti-Ge3, and 1 alloanti-Ge4. Titrations revealed weak expression of these antigens on her RBCs and those of her compatible siblings as compared with controls. In contrast, titrations of mouse and rat monoclonal antibodies specific for GPC/GPD showed no differences. Western blotting of RBC membranes using GPC/GPD specific monoclonal antibodies showed a broad diffuse band corresponding to GPC.Ge in addition to GPC and GPD. Blotting of membranes from trypsin-treated RBCs from these individuals revealed an increase of 1500 in M(r) of membrane-bound tryptic fragment over that in the membranes from typsin-treated RBCs from persons with normal GPC/GPD. In RT-PCR, two products were obtained for RW and her compatible siblings: one had a complete deletion of exon 3 and the other had a base change (A-->T) in nucleotide 173 in exon 3 (confirmed by genomic DNA sequencing of exon 3). This point mutation has resulted in the loss of restriction enzyme Tth111 I-sensitive site in the mutant GYPC. CONCLUSION: The specificity of antibody in RW's serum was serologically anti-Ge2. Two genetic events occurred in exon 3 in GYPC of RW and her compatible siblings. The exon 3 deletion confirmed a Ge:-2,-3,4 haplotype. The abnormal tryptic fragment obtained was due to the (A173-->T) base change in exon 3 that resulted in Asp58-->Val in the deduced amino acid sequence at the membrane boundary.

Animals↗

PubMedDate not supplied

Genotyping of RHD by multiplex polymerase chain reaction analysis of six RHD-specific exons.

BACKGROUND: Qualitative RHD variants are the result of the replacement of RHD exons by their RHCE counterparts or of point mutations in RHD causing amino acid substitutions. For RHD typing, the use of at least two RHD typing polymerase chain reaction (PCR) assays directed at different regions of RHD is advised to prevent discrepancies between phenotyping and genotyping results, but even then discrepancies occur. A multiplex RHD PCR based on amplification of six RHD-specific exons in one reaction mixture is described. STUDY DESIGN AND METHODS: Six RHD-specific primer sets were designed to amplify RHD exons 3, 4, 5, 6, 7, and 9. DNA from 119 donors (87 D+, 14 D- and 18 with known D variants; whites and nonwhites) with known Rh phenotypes was analyzed. RESULTS: All six RHD-specific exons from 85 D+ individuals were amplified, whereas none of the RHD exons from 13 D- individuals were amplified. Multiplex PCR analysis showed that the genotypes of two donors typed as D+ were DIVa and DVa. Red cell typing confirmed these findings. From all D variants tested (DIIIc, DIVa, DIVb, DVa, DVI, DDFR, DDBT) and from RoHar, RHD-specific exons were amplified as expected from the proposed genotypes. CONCLUSION: The multiplex PCR assay is reliable in determining genotypes in people who have the D+ and partial D phenotypes as well as in discovering people with new D variants. Because the multiplex PCR is directed at six regions of RHD, the chance of discrepancies is markedly reduced. The entire analysis can be performed in one reaction mixture, which results in higher speed, higher accuracy, and the need for smaller samples. This technique might be of great value in prenatal RHD genotyping.

Alleles↗

PubMed2000

Htra2-beta 1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 (SMN2).

Spinal muscular atrophy (SMA), a common motor neuron disease in humans, results from loss of functional survival motor neuron (SMN1) alleles. A nearly identical copy of the gene, SMN2, fails to provide protection from SMA because of a single translationally silent nucleotide difference in exon 7. This likely disrupts an exonic splicing enhancer and causes exon 7 skipping, leading to abundant production of a shorter isoform, SMN2Delta7. The truncated transcript encodes a less stable protein with reduced self-oligomerization activity that fails to compensate for the loss of SMN1. This report describes the identification of an in vivo regulator of SMN mRNA processing. Htra2-beta1, an SR-like splicing factor and ortholog of Drosophila melanogaster transformer-2, promoted the inclusion of SMN exon 7, which would stimulate full-length SMN2 expression. Htra2-beta1 specifically functioned through and bound an AG-rich exonic splicing enhancer in SMN exon 7. This effect is not species-specific as expression of Htra2-beta1 in human or mouse cells carrying an SMN2 minigene dramatically increased production of full-length SMN2. This demonstrates that SMN2 mRNA processing can be modulated in vivo. Because all SMA patients retain at least one SMN2 copy, these results show that an in vivo modulation of SMN RNA processing could serve as a therapeutic strategy to prevent SMA.

Animals↗

PubMed1986

Individual exons encode the integral membrane domains of human myelin proteolipid protein.

The gene encoding human proteolipid protein (PLP) was isolated from a human genomic library by hybridization with labeled DNA of a PLP-specific cDNA clone. The entire PLP gene spans approximately 17 kilobases. Restriction and sequence analysis revealed seven exons and six introns. The entire nucleotide sequences of the exons and of the exon-intron transitions were determined, and the intron lengths were measured. Exon I includes only ATGG of the translated region, the N-terminal methionine codon and G of glycine, the first amino acid of mature PLP. Each hydrophobic trans- and cis-membrane domain of PLP together with its adjacent hydrophilic sequence correlates closely with one exon of the gene except for the C-terminal transmembrane helix that is encoded by two exons. The amino acid sequence of human PLP derived from the nucleic acid sequence is highly conserved. Human and rat PLP are completely homologous, whereas only four amino acid residues are exchanged in bovine PLP sequence derived from protein sequencing and a partial cDNA clone. Homology search on the nucleic acid level among human, bovine, and rat brain PLPs indicates an unusually high homology in the coding regions. Hybridization analysis with DNA of human-rodent hybrid clones revealed that the gene encoding PLP segregates with human X chromosome in the region q13-q22.

Amino Acid Sequence↗

PubMed1988

Gene structure for the alpha 1 chain of a human short-chain collagen (type XIII) with alternatively spliced transcripts and translation termination codon at the 5' end of the last exon.

Two overlapping human genomic clones that encode a short-chain collagen, designated alpha 1(XIII), were isolated by using recently described cDNA clones. Characterization of the cosmid clones that span approximately equal to 65,000 base pairs (bp) of the 3' end of the gene established several unusual features of this collagen gene. The last exon encodes solely the 3' untranslated region and it begins with a complete stop codon. The 10 adjacent exons vary in size from 27 to 87 bp and two of them are 54 bp. Therefore, the alpha 1-chain gene of type XIII collagen has some features found in genes for fibrillar collagens but other features that are distinctly different. Previous analysis of overlapping cDNA clones and nuclease S1 mapping of mRNAs indicated one alternative splicing site causing a deletion of 36 bp from the mature mRNA. The present study showed that the 36 bp is contained within the gene as a single exon and also that the gene has a 45-bp -Gly-Xaa-Xaa- repeat coding exon not found in the cDNA clones previously characterized. Nuclease S1 mapping experiments indicated that this 45-bp exon is found in normal human skin fibroblast mRNAs. Accordingly, the data demonstrate that there is alternative splicing of at least two exons of the type alpha 1(XIII)-chain gene.

Base Sequence↗

PubMed1989

Reordering of nine exons is necessary to form a functional actin gene in Oxytricha nova.

During the development of a macronucleus from a micronucleus after cell mating in hypotrichs all the genes (approximately 20,000) are excised from micronuclear chromosomes as individual small DNA molecules. Telomeres are added to the ends of each gene-sized molecule and each is amplified, mostly by approximately 1000-fold, to yield a transcriptionally active macronucleus. As a part of the study of the excision of genes from chromosomes, we have cloned six fragments of chromosomal DNA from Oxytricha nova, each containing a full copy of an actin gene, for comparison with the structure of the actin-encoding DNA molecule in the macronucleus. All six micronuclear actin clones had the same overall organization as judged by restriction mapping. Two micronuclear actin clones were sequenced. These differ from one another at a few nucleotide positions but both prescribe precisely the same actin polypeptide. Both micronuclear actin genes contain nine exons separated by eight intron-like sequences. The macronuclear gene contains these nine exons without intron-like segments. Assigning the order 1 through 9 to the nine micronuclear exons, the order in the macronucleus is 8-7-1-2-4-3-5-9-6. In the micronuclear actin gene, all nine exons possess terminal repeat sequences. These repeat sequences provide precise directions for reordering and joining of the nine exons to yield the exon order in the macronuclear gene. Polymerase chain reaction analysis of micronuclear DNA of the related species, Oxytricha trifallax, shows that the actin gene has an unorthodox arrangement in this species also.

Actins↗

PubMed1990

Splicing of COB intron 5 requires pairing between the internal guide sequence and both flanking exons.

Group I introns are characterized by a set of conserved sequence elements and secondary structures. Evidence supporting the pairing of certain of these sequences has come from the comparison of intron sequences and from the analysis of mutations that disrupt splicing by interfering with pairing. One of the structures proposed for all group I introns is an internal guide sequence that base pairs with the upstream and the downstream exons, bringing them into alignment for ligation. We made specific mutations in the internal guide sequence and the flanking exons of the fifth intron in the yeast mitochondrial gene for apocytochrome b (COB). Mutations that disrupted the pairing between the internal guide sequence and the upstream exon (the P1 pairing) blocked addition of guanosine to the 5' end of the intron during autocatalytic reactions and prevented formation of the full-length circular intron. In contrast, transcripts containing mutations that disrupted the pairing between the guide sequence and the downstream exon (the P10 helix) initiated splicing but failed to ligate exons. Compensatory mutations that restored helices of normal stability mitigated the effects of the original mutations. These data provide direct evidence for the importance of the base pairing between the internal guide sequence and the downstream exon in the splicing of a wild-type group I intron.

Base Composition↗

PubMed1999

Evidence for the function of an exonic splicing enhancer after the first catalytic step of pre-mRNA splicing.

Exonic splicing enhancers (ESEs) activate pre-mRNA splicing by promoting the use of the flanking splice sites. They are recognized by members of the serine/arginine-rich (SR) family of proteins, such as splicing factor 2/alternative splicing factor (SF2/ASF), which recruit basal splicing factors to form the initial complexes during spliceosome assembly. The in vitro splicing kinetics of an ESE-dependent IgM pre-mRNA suggested that an SF2/ASF-specific ESE has additional functions later in the splicing reaction, after the completion of the first catalytic step. A bimolecular exon ligation assay, which physically uncouples the first and second catalytic steps of splicing in a trans-splicing reaction, was adapted to test the function of the ESE after the first step. A 3' exon containing the SF2/ASF-specific ESE underwent bimolecular exon ligation, whereas 3' exons without the ESE or with control sequences did not. The ESE-dependent trans-splicing reaction occurred after inactivation of U1 or U2 small nuclear ribonucleoprotein particles, compatible with a functional assay for events after the first step of splicing. The ESE-dependent step appears to take place before the ATP-independent part of the second catalytic step. Bimolecular exon ligation also occurred in an S100 cytosolic extract, requiring both the SF2/ASF-dependent ESE and complementation with SF2/ASF. These data suggest that some ESEs can act late in the splicing reaction, together with appropriate SR proteins, to enhance the second catalytic step of splicing.

Adenosine Triphosphate↗

PubMed1996

Mapping the functional domains within the carboxyl terminus of alpha-tropomyosin encoded by the alternatively spliced ninth exon.

Tropomyosins are highly conserved, coiled-coil actin binding proteins found in most eukaryotic cells. Striated and smooth muscle alpha-tropomyosins differ by the regions encoded by exons 2 and 9. Unacetylated smooth tropomyosin expressed in Escherichia coli binds actin with high affinity, whereas unacetylated striated tropomyosin requires troponin, found only in striated muscle, for strong actin binding. The residues encoded by exon 9 cause these differences (Cho, Y.-J., and Hitchcock-DeGregori, S. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10153-10157). We mapped the functional domains encoded by the alpha-tropomyosin exon 9a (striated muscle-specific) and 9d (constitutively expressed), by measuring actin binding and regulation of the actomyosin MgATPase by tropomyosin exon 9 chimeras and truncation mutants expressed in E. coli. We have shown that: 1) the carboxyl-terminal nine residues define the actin affinity of unacetylated tropomyosin; 2) in the presence of Ca2+, the entire exon 9a is required for troponin to promote fully high affinity actin binding; 3) the first 18 residues encoded by exon 9a are critical for the interaction of troponin with tropomyosin on the thin filament, even in the absence of Ca2+. The results give new insight into the structural requirements of tropomyosin for thin filament assembly and regulatory function.

Actomyosin↗

PubMed1997

A regulatory element within a coding exon modulates keratin 18 gene expression in transgenic mice.

Multiple tissue-specific, DNase-hypersensitive sites are correlated with known or potential regulatory regions of the human keratin 18 (K18) gene. One of these sites is found within exon 6, close to a potential AP-1 binding site. Footprint analysis confirmed that this site is capable of binding c-Jun and c-Fos in vitro. However, exon 6 can stimulate expression of a reporter gene driven by the K18 proximal promoter independent of AP-1 in F9 cells and additionally modulates AP-1 responsiveness when in combination with an intron enhancer. Analysis in transgenic mice and by transient transfections of mutant forms of the K18 gene showed that exon 6 contributes to the expression of the K18 gene. However, substitution of part of exon 6 with the corresponding part of the keratin 19 gene which lacks an AP-1 site decreased but did not destroy the regulatory activity of the exon. Furthermore, this mutation did not alter either the tissue specificity or the position-independent and copy number-dependent behavior of the K18 gene. In contrast, a frameshift mutation within exon 6 dramatically decreased the expression of the gene. K18 RNA expression from the frameshift mutation was less than 10% of the wild type K18 transgene. This decline in expression was the result of a combination of decreased stability of mutant K18 RNA and the creation of a negative regulatory element that can interact with the first intron regulatory elements and actively suppress K18 expression. These results demonstrate that a protein-coding portion of the K18 gene also has a regulatory function.

Amino Acid Sequence↗

PubMed1999

5' splice site mutations in tau associated with the inherited dementia FTDP-17 affect a stem-loop structure that regulates alternative splicing of exon 10.

Missense and splice site mutations in the microtubule-associated protein tau gene were recently found associated with fronto-temporal dementia and parkinsonism linked to chromosome 17 (Poorkaj et al. (1998) Ann. Neurol. 43, 815-825; Hutton et al. (1998) Nature 393, 702-705; Spillantini et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7737-7741). The mutations in the 5' splice site of exon 10 were shown to increase the ratio of tau mRNAs containing exon 10 and thus the proportion of Tau protein isoforms with 4 microtubule binding repeat domains, although how this increase leads to neurodegeneration is presently unclear. The mechanism by which these mutations increase tau exon 10 splicing was not determined, although the mutations were predicted to disrupt a potential stem-loop structure that was likely involved in the regulation of exon 10 alternative splicing. Here we describe in vitro splicing assays and RNA structural analysis that demonstrate that the mutations do indeed act through disruption of the stem-loop structure and that the stability of this secondary structure feature at least partially determines the ratio of tau exon 10+/- transcripts. In addition, we provide evidence that the stability of the stem-loop structure underlies the alternative splicing of this exon in other species.

Alternative Splicing↗