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PubMed1998

Complete exon-intron organization of the human gene for the alpha1 chain of type XV collagen (COL15A1) and comparison with the homologous COL18A1 gene.

The human gene for the alpha1 chain of type XV collagen (COL15A1) is about 145 kilobases in size and contains 42 exons. The promoter is characterized by the lack of a TATAA motif and the presence of several Sp1 binding sites, some of which appeared to be functional in transfected HeLa cells. Comparison with Col18a1, which encodes the alpha1(XVIII) collagen chain homologous with alpha1(XV), indicates marked structural homology spread throughout the two genes. The mouse Col18a1 contains one exon more than COL15A1, due to the fact that COL15A1 lacks sequences corresponding to exon 3 of Col18a1, which encodes a cysteine-rich sequence motif. Twenty-five of the exons of the two genes are almost identical in size, six of them contain conserved split codons, and the locations of the respective exon-intron junctions are identical or almost identical in the two genes. The homologous exons include the closely adjacent first pair of exons and the exons encoding a thrombospondin-1 homology found in the N-terminal noncollagenous domain 1, which are followed by the most variable part of the two genes, covering the C-terminal half of their noncollagenous domain 1 and the beginning of the collagenous portion, after which most of the exons are homologous. The lengths of the introns are not similar in these genes, with two exceptions, namely the first intron, which is very short, less than 100 base pairs, and the second intron, which is very large, about 50 kilobases, in both genes. It can be concluded that COL15A1 and Col18a1 are derived from a common ancestor.

Adult↗

PubMed2003

The CD44 alternative v9 exon contains a splicing enhancer responsive to the SR proteins 9G8, ASF/SF2, and SRp20.

The CD44 gene alternative exons v8, v9, and v10 are frequently spliced as a block by epithelial cells. By transfecting minigenes containing only one of these alternative exons, we show that splicing of each of them is under cell type-specific control. By using minigenes carrying short block mutations within exons v8 and v9, we detected a candidate exon splicing enhancer in each of these exons. These candidates activated splicing in vitro of a heterologous transcript and are thus true exon splicing enhancers. We analyzed further a v9 exon splicing enhancer covering approximately 30 nucleotides. This enhancer can be UV cross-linked to SR proteins of 35 and 20 kDa in HeLa nuclear extract. By using individual recombinant SR proteins for UV cross-linking in S100 extract, these proteins were identified as 9G8, ASF/SF2, and SRp20. S100 complementation studies using recombinant 9G8, ASF/SF2, and SRp20 showed that all three proteins can activate splicing in vitro of a heterologous exon containing the v9 enhancer; the strongest activation was obtained with 9G8. Progressive truncation of the 30-nucleotide enhancer leads to a progressive decrease in splicing activation. We propose that 9G8, ASF/SF2, SRp20, and possibly other non-SR proteins cooperate in vivo to activate v9 exon splicing.

Alternative Splicing↗

PubMed1998

Nearby stop codons in exons of the neurofibromatosis type 1 gene are disparate splice effectors.

Stop mutations are known to disrupt gene function in different ways. They both give rise to truncated polypeptides because of the premature-termination codons (PTCs) and frequently affect the metabolism of the corresponding mRNAs. The analysis of neurofibromin transcripts from different neurofibromatosis type 1 (NF1) patients revealed the skipping of exons containing PTCs. The phenomenon of exon skipping induced by nonsense mutations has been described for other disease genes, including the CFTR (cystic fibrosis transmembrance conductance regulator) gene and the fibrillin gene. We characterized several stop mutations localized within a few base pairs in exons 7 and 37 and noticed complete skipping of either exon in some cases. Because skipping of exon 7 and of exon 37 does not lead to a frameshift, PTCs are avoided in that way. Nuclear-scanning mechanisms for PTCs have been postulated to trigger the removal of the affected exons from the transcript. However, other stop mutations that we found in either NF1 exon did not lead to a skip, although they were localized within the same region. Calculations of minimum-free-energy structures of the respective regions suggest that both changes in the secondary structure of the mRNA and creation or disruption of exonic sequences relevant for the splicing process might in fact cause these different splice phenomena observed in the NF1 gene.

Alternative Splicing↗

PubMed1995

Function of exon 2 in optimal trans-activation by Tat of HIV type 2.

HIV-1 and HIV-2 are human retroviruses whose life cycles require viral regulatory proteins, one of which is the trans-activator, Tat. Tat of HIV-1 (Tat-1) displays modular function with independent activation function localized to the amino-terminal, cysteine-rich, and core regions and independent RNA-binding function localized to a basic region. These functional domains are contained in the first of two exons encoding Tat-1; deletion of exon 2 does not contribute to functional domains of Tat-1. Tat of HIV-2 (Tat-2) has structurally analogous regions, but the amino terminus, basic region, and carboxy terminus encoded by exon 2 display amino acid sequence and functional divergence compared to Tat-1. We have shown that, in contrast to Tat-1, exon 2 of Tat-2 (residues 100 to 130) is required for optimal trans-activation of HIV-1 and HIV-2 long terminal repeats (LTRs). Here we demonstrate that a series of basic residues in exon 2 are required for these effects. Exon 2 does not alter the level of protein expression of Tat-2. Further, in the context of heterologous DNA binding, exon 2 does not contribute to activation function. These data suggest that full-length Tat-2 results in optimal trans-activation through enhanced RNA-binding function of exon 1 by involvement of a basic region in exon 2. Differential expression of short and full-length Tats during different stages of the HIV-2 life cycle might regulate levels of viral expression, viral replication, and resultant cytopathology.

Amino Acid Sequence↗

PubMed2001

Positional dissociation between the genetic mutation responsible for pseudohypoparathyroidism type Ib and the associated methylation defect at exon A/B: evidence for a long-range regulatory element within the imprinted GNAS1 locus.

Pseudohypoparathyroidism type Ib (PHP-Ib) is a paternally imprinted disorder which maps to a region on chromosome 20q13.3 that comprises GNAS1 at its telomeric boundary. Exon A/B of this gene was recently shown to display a loss of methylation in several PHP-Ib patients. In nine unrelated PHP-Ib kindreds, in whom haplotype analysis and mode of inheritance provided no evidence against linkage to this chromosomal region, we confirmed lack of exon A/B methylation for affected individuals, while unaffected carriers showed no epigenetic abnormality at this locus. However, affected individuals in one kindred (Y2) displayed additional methylation defects involving exons NESP55, AS and XL, and unaffected carriers in this family showed an abnormal methylation at exon NESP55, but not at other exons. Taken together, current evidence thus suggests that distinct mutations within or close to GNAS1 can lead to PHP-Ib and the associated epigenetic changes. To further delineate the telomeric boundary of the PHP-Ib locus, the previously reported kindred F, in which patient F-V/51 is recombinant within GNAS1, was investigated with several new markers and direct nucleotide sequence analysis. These studies revealed that F-V/51 remains recombinant at a single nucleotide polymorphism (SNP) located 1.2 kb upstream of XL. No heterozygous mutation was identified between exon XL and an SNP approximately 8 kb upstream of NESP55, where this affected individual becomes linked, suggesting that the genetic defect responsible for parathyroid hormone resistance in kindred F, and probably other PHP-Ib patients, is located >or=56 kb centromeric of the abnormally methylated exon A/B. A region upstream of the known coding exons of GNAS1 is therefore predicted to exert, presumably through imprinting of exon A/B, long-range effects on G(s)alpha expression.

Chromosome Mapping↗

PubMed1999

Functional analysis of cis-acting elements regulating the alternative splicing of human CFTR exon 9.

The rate of exon 9 exclusion from the cystic fibrosis transmembrane conductance regulator (CFTR) mRNA is associated with monosymptomatic forms of cystic fibrosis. Exon 9 alternative splicing is modulated by a polymorphic polythymidine tract within its 3' splice site. We have generated a minigene carrying human CFTR exon 9 with its flanking intronic sequences and set up an in vivo model to study the cis-acting DNA elements which modulate its splicing. Transfections into human cell lines showed that T5, but not T9 or T7 alleles, significantly increases the alternative splicing of exon 9. Moreover, we found that another polymorphic locus juxtaposed upstream of the T tract, and constituted by (TG)(n)repeats, can further modulate exon 9 skipping but only when activated by the T5 allele. Then, we extended our studies to the mouse CFTR exon 9 which does not show alternative splicing. Comparison of human and mouse introns 8 and 9 revealed a low homology between the two sequences and the absence of the human polymorphic loci within the mouse intron 3' splice site. We have tested a series of constructs where the whole human exon 9 with its flanking intronic sequences was replaced partially or completely by the murine counterpart. The transfections of these constructs in human and murine cell lines reveal that also sequences of the downstream intron 9 affect exon 9 definition and co-modulate, with the UG/U 3' splice site sequences, the extent of exon 9 skipping in CFTR mRNA.

Alternative Splicing↗

PubMed1995

Drug-responsive and tissue-specific alternative expression of multiple first exons in rat UDP-glucuronosyltransferase family 1 (UGT1) gene complex.

Genomic clones of UDP-glucuronosyltransferase family 1 (UGT1) were isolated from wild-type Wistar rats. The UGT1 locus spans > 120 kb and forms a gene complex. In this locus nine unique first exons encoding NH2-terminal portions of each isoform were located at intervals of approximately 10 kb and followed by only one set of commonly used exons (exons II, III, IV, and V) encoding the COOH-terminal portion. From sequence analyses of the unique first exons, the amino acid sequences of the isoforms were deduced and they were divided into two groups: the Bilirubin cluster (B cluster) and the Phenol cluster (A cluster). A and B clusters consisted of four (A1-A4) and five (B1-B5) isoform-specific exons, respectively. A2, A3, B3, and B4 were identified as previously uncharacterized forms, while A4 and B4 were pseudogenes. Isoform B1 was a major component in hepatic microsomes of untreated rats and was induced in clofibrate- and dexamethasone-administered rats. A slight but a significant amount of B1 mRNA was also detected in various tissues such as intestine. mRNAs coding for isoform A1 and isoform A2 were induced in livers of methylcholanthrene (MC)-treated rats. Induction of A1 mRNA was also observed in kidneys of MC-treated rats. A genomic clone containing the commonly used exons was also isolated from Gunn rats and a single base deletion was identified in exon IV. Isoforms of the UGT1 family are made from the complex gene locus by an alternative combination of one of the unique first exons with the commonly used exons.

Alternative Splicing↗

PubMed1988

Intron sequences and the length of the downstream second exon affect the binding of hnRNP C proteins in an in vitro splicing reaction.

The proteins that are in direct contact with the pre-mRNA in an in vitro splicing reaction were analyzed by UV cross-linking experiments. Six major proteins (120, 55, 44, 42, 39 and 38 KD) and three minor polypeptides (84, 72 and 63 KD) were detected. The predominant proteins 44, 42 KD belong to the class of hnRNP C proteins since they were immunoprecipitated by monoclonal antibodies directed against hnRNP C proteins. The cross-linked proteins were not detected in the absence of Mg2+, ATP or when RNA lacking introns were used as substrates in the splicing reactions. The effect of exon sequences on the binding efficiency for the photocrosslinked proteins was investigated. Transcripts containing a second exon of 24 nucleotides for the beta-globin or 107 nucleotides for the mouse insulin, yielded a reduced amount of cross-linked proteins when compared with "full length" pre-mRNAs. Sequences within the first exon of the beta-globin pre-mRNA did not affect the binding efficiency of these proteins. The reduced binding efficiency of the cross-linked proteins for the truncated beta-globin or mouse insulin pre-mRNAs correlated with the lower efficiency for in vitro splicing. Substitutions with unrelated sequences in the beta-globin second exon restore the binding of the cross-linked proteins indicating that the length of the second exon and not specific sequences are relevant for the binding efficiency of these proteins. The SP6/mouse insulin oligonucleotides cross-linked to the hnRNP C proteins were isolated and sequenced. A 17-mer was located in the second exon (134 nucleotides downstream from the 3' splice site) and a 14-mer in the intron region (25 nucleotides downstream the 5' splice site). The beta-globin oligonucleotides cross-linked to the hnRNP C proteins were a 13-mer in the second exon (28 nucleotides downstream the 3' splice site) and an 8-mer in the first exon (81 nucleotides downstream the 5' end of the pre-mRNA). Our results indicate that the hnRNP C proteins interact with those oligonucleotides located in different regions of the pre-mRNA. The binding efficiency of those proteins, however, depends on the length of the second exon and the presence of intron sequences (secondary and/or tertiary pre-mRNA structure).

Adenosine Triphosphate↗

PubMed1999

An element in the 5' common exon of the NCAM alternative splicing unit interacts with SR proteins and modulates 5' splice site selection.

The neural cell adhesion molecule (NCAM) gene contains an 801 nt exon that is included preferentially in neuronal cells. We have set up an in vitro splicing system that mimics the neuro-specific alternative splicing profile of NCAM exon 18. Splicing regulation is observed using model pre-mRNAs that contain competing 5' or 3' splice sites, suggesting that distinct pathways regulate NCAM 5' and 3' splice site selection. While inclusion of exon 18 is the predom-inant choice in neuronal cells, an element in the 5' common exon 17 improves exon 17/exon 19 splicing in a neuronal cell line. A similar behavior is observed in vitro as the element can stimulate the 5' splice site of exon 17 or a heterologous 5' splice site. The minimal 32 nt sequence of the exon 17 enhancer consists of purine stretches and A/C motifs. Mutations in the purine stretches compromise the binding of SR proteins and decreases splicing stimulation in vitro. Mutations in the A/C motifs do not affect SR protein binding but reduce enhancing activity. Our results suggest that the assembly of an enhancer complex containing SR proteins in a 5' common exon ensures that NCAM mRNAs lacking exon 18 are made in neuronal cells.

Alternative Splicing↗

PubMed2006

Reduced splicing efficiency induced by synonymous substitutions may generate a substrate for natural selection of new splicing isoforms: the case of CFTR exon 12.

Alternative splicing has been associated with increased evolutionary changes and with recent exon creation or loss. The addition of a new exon can be explained by its inclusion in only a fraction of the transcripts leaving the original form intact and giving to the new form the possibility to evolve independently but the exon loss phenomenon is less clear. To explore the mechanism that could be involved in CFTR exon 12 lower splicing efficiency in primates, we have analyzed the effect of multiple synonymous variations. Random patterns of synonymous variations were created in CFTR exon12 and the majority of them induced exon inclusion, suggesting a suboptimal splicing efficiency of the human gene. In addition, the effect of each single synonymous substitution on splicing is strongly dependent on the exonic context and does not correlate with available in silico exon splicing prediction programs. We propose that casual synonymous substitutions may lead to a reduced splicing efficiency that can result in a variable proportion of exon loss. If this phenomenon happens in in-frame exons and to an extent tolerated by the cells it can have an important evolutionary effect since it may generate a substrate for natural selection of new splicing isoforms.

Alternative Splicing↗

PubMed2001

Intergenic transcripts containing a novel human cytochrome P450 2C exon 1 spliced to sequences from the CYP2C9 gene.

The cytochrome P450 2C (CYP2C) gene locus was found to include a novel exon 1 sequence with high similarity to the canonical exon 1 of CYP2C18. Rapid amplification of cDNA ends (RACE) and PCR amplifications of human liver cDNA revealed the presence of several intergenic species containing the CYP2C18 exon 1-like sequence spliced to different combinations of exonic and intronic sequences from the CYP2C9 gene. One splice variant was found to have an open reading frame starting at the canonical translation initiation codon of the CYP2C18 exon 1-like sequence. Another variant consisted of the nine typical CYP2C9 exons spliced after the CYP2C18 exon 1-like sequence through a segment of CYP2C9 5' flanking sequences. Moreover, analysis of bacterial artificial chromosome (BAC) clones revealed that the CYP2C18 exon 1-like sequence was located in the intergenic region between the CYP2C19 and CYP2C9 genes. The finding that a solitary exon is spliced with sequences from a neighboring gene may be interpreted as representing a general evolutionary mechanism aimed at using the full expression potential of a cell's genomic informational content.

5' Flanking Region↗

PubMed2002

The 5' leader of plant PgiC has an intron: the leader shows both the loss and maintenance of constraints compared with introns and exons in the coding region.

PgiC, a complex gene with 23 coding exons and 22 intervening introns, encodes the cytosolic isozyme of phosphoglucose isomerase (EC 5.3.1.9) in higher plants. Here, we report RNA ligase-mediated rapid amplification of cDNA ends experiments that showed that PgiC in Clarkia (Onagraceae) and Arabidopsis thaliana has an intron in the 5' leader. Comparison of the EMBL accessions of the cDNA and genomic sequences showed that this is also the case in rice (Oryza sativa), suggesting that a leader intron is generally present in higher plant PgiC. The intron is bounded by consensus 5'-GT and AG-3' splice sites but showed alternative splicing in Clarkia, resulting in mature transcripts that differ by 8-19 nt in length. The intron is located 18 or 10 nt upstream of the start codon in Clarkia, 2 nt upstream in Arabidopsis, and 9 nt in rice. PgiC in Clarkia was duplicated before the divergence of the extant species, many of which have two expressed genes PgiC1 and PgiC2. Full-length transcripts of both genes identified the transcription start and made it possible to identify the leader intron and leader exon (between the transcription start and leader intron) from previously obtained genomic sequences of both genes in other Clarkia species. These data permit the comparison of evolution in the leader exon and intron with the exons and introns of the coding region, a topic that has not been studied previously. Both the leader exon and the leader intron resemble introns of the coding region in base substitution rate and accumulation of gaps. But the leader intron splice junctions are not strictly conserved in position as are those of the coding region introns. Also, in base composition, the leader intron resembles the other introns, whereas the leader exon more nearly resembles the coding exons. A difference in base composition between coding exons and flanking introns is known to be important for the recognition of splice sites. Thus, the marked difference in base composition between the leader exon and leader intron is probably maintained by selection despite a high rate of sequence divergence.

5' Untranslated Regions↗

PubMed1999

Polymorphism across an exon-intron boundary in an avian Mhc class II B gene.

Twenty-three sequence haplotypes spanning the boundary of the second exon and intron of a red-winged blackbird Mhc class II B gene, Agph-DAB1, are presented. The polymorphism of the exon segment is distributed in two divergent allelic lineages which appear to be maintained by balancing selection. The silent nucleotide diversity of the exon (pi = 0.101) is more than five times that of the intron (pi = 0.018) and decays rapidly across the exon-intron boundary. Additionally, genealogical reconstruction indicates that divergence from a common ancestor in the exon sample is over four times that of the intron. The intron sequences reveal a pattern of polymorphism which is characteristic of directional selection, rather than a pattern expected from linkage to a balanced polymorphism. These results suggest that the evolutionary histories of these two adjacent regions have been disassociated by recombination or gene conversion. The estimated population recombination parameter between the exon and the intron is sufficiently high (4NeC = 8.545) to explain the homogenization of intron sequences. Compatibility analyses estimate that these events primarily occur from the exon-intron boundary to about 20-30 bases into the intron. Additionally, the observation that divergent exon alleles share identical intron sequence supports the conclusion of disassociation of exon and intron evolutionary histories by recombination.

Amino Acid Sequence↗

PubMed2003

Multiple variable first exons: a mechanism for cell- and tissue-specific gene regulation.

A large family of neural protocadherin (Pcdh) proteins is encoded by three closely linked mammalian gene clusters (alpha, beta, and gamma). Pcdh alpha and gamma clusters have a striking genomic organization. Specifically, each "variable" exon is spliced to a common set of downstream "constant" exons within each cluster. Recent studies demonstrated that the cell-specific expression of each Pcdh gene is determined bya combination of variable-exon promoter activation and cis-splicing of the corresponding variable exon to the first constant exon. To determine whether there are other similarly organized gene clusters in mammalian genomes, we performed a genome-wide search and identified a large number of mammalian genes containing multiple variable first exons. Here we describe several clusters that contain about a dozen variable exons arrayed in tandem, including UDP glucuronosyltransferase (UGT1), plectin, neuronal nitric oxide synthase (NOS1), and glucocorticoid receptor (GR) genes. In all these cases, multiple variable first exons are each spliced to a common set of downstream constant exons to generate diverse functional mRNAs. As an example, we analyzed the tissue-specific expression profile of the mouse UGT1 repertoire and found that multiple isoforms are expressed in a tissue-specific manner. Therefore, this variable and constant genomic organization provides a genetic mechanism for directing distinct cell- and tissue-specific patterns of gene expression.

Animals↗

PubMed2006

Evolution of exon-intron structure and alternative splicing in fruit flies and malarial mosquito genomes.

Comparative analysis of alternative splicing of orthologous genes from fruit flies (Drosophila melanogaster and Drosophila pseudoobscura) and mosquito (Anopheles gambiae) demonstrated that both in the fruit fly genes and in fruit fly-mosquito comparisons, constitutive exons and splicing sites are more conserved than alternative ones. While >97% of constitutive D. melanogaster exons are conserved in D. pseudoobscura, only approximately 80% of alternative exons are conserved. Similarly, 77% of constitutive fruit fly exons are conserved in the mosquito genes, compared with <50% of alternative exons. Internal alternatives are more conserved than terminal ones. Retained introns are the least conserved, alternative acceptor sites are slightly more conserved than donor sites, and mutually exclusive exons are almost as conserved as constitutive exons. Cassette and mutually exclusive exons experience almost no intron insertions. We also observed cases of interconversion of various elementary alternatives, e.g., transformation of cassette exons into alternative sites. These results agree with the observations made earlier in human-mouse comparisons and demonstrate that the phenomenon of relatively low conservation of alternatively spliced regions may be universal, as it has been observed in different taxonomic groups (mammals and insects) and at various evolutionary distances.

Alternative Splicing↗

PubMed1992

Chicken vigilin gene organization and expression pattern. The domain structure of the protein is reflected by the exon structure.

Chicken vigilin was identified as a member of an evolutionary-conserved protein family with a unique repetitive domain structure. 14 tandemly repeated domains are found in chicken vigilin, all of which consist of a conserved sequence motif (subdomain A) and a potential alpha-helical region (subdomain B) [1]. We have established the physical structure of the chicken vigilin gene by restriction-fragment analysis and DNA sequencing of overlapping clones isolated from a phage lambda genomic DNA library. The chicken vigilin gene is a single-copy gene with a total of 27 exons which are distributed over a region of some 22 kbp. Exon 1 codes for a portion of the 5' untranslated region, exon 2 contains the translation start point and forms, along with exons 3 and 4, the N-terminal non-domain region. Exons 5-25 encode the vigilin domains 1-14 and the remaining exons 26 and 27 contain the non-domain C-terminal as well as the untranslated regions. The domain structure of the protein is reflected in the positioning of introns which demarcate individual domains. While domains 1-3 and 8-10 are each encoded by a single exon (5-7, 16-18); all other domains are contained in a set of two exons which are separated by introns interspersed at variable positions of the DNA segment coding for the conserved sequence motif. In conclusion, the data presented suggest that the chicken vigilin gene evolved by amplification of a primordial exon unit coding for the fundamental bipartite vigilin domain.

Animals↗

PubMed2006

Thermal unfolding of smooth muscle and nonmuscle tropomyosin alpha-homodimers with alternatively spliced exons.

We used differential scanning calorimetry (DSC) and circular dichroism (CD) to investigate thermal unfolding of recombinant fibroblast isoforms of alpha-tropomyosin (Tm) in comparison with that of smooth muscle Tm. These two nonmuscle Tm isoforms 5a and 5b differ internally only by exons 6b/6a, and they both differ from smooth muscle Tm by the N-terminal exon 1b which replaces the muscle-specific exons 1a and 2a. We show that the presence of exon 1b dramatically decreases the measurable calorimetric enthalpy of the thermal unfolding of Tm observed with DSC, although it has no influence on the alpha-helix content of Tm or on the end-to-end interaction between Tm dimers. The results suggest that a significant part of the molecule of fibroblast Tm (but not smooth muscle Tm) unfolds noncooperatively, with the enthalpy no longer visible in the cooperative thermal transitions measured. On the other hand, both DSC and CD studies show that replacement of muscle exons 1a and 2a by nonmuscle exon 1b not only increases the thermal stability of the N-terminal part of Tm, but also significantly stabilizes Tm by shifting the major thermal transition of Tm to higher temperature. Replacement of exon 6b by exon 6a leads to additional increase in the alpha-Tm thermal stability. Thus, our data show for the first time a significant difference in the thermal unfolding between muscle and nonmuscle alpha-Tm isoforms, and indicate that replacement of alternatively spliced exons alters the stability of the entire Tm molecule.

Actins↗

PubMed1993

Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF.

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.

Animals↗