Multiple sites of alternative splicing of the rat fibronectin gene transcript

We describe analyses of the structure and expression of the rat fibronectin gene with particular attention to the 40-kb stretch from the center of the gene which encodes 17 type-III repeating units. Each repeat is precisely separated from its neighbors by introns and most are encoded by pairs of exons. Three repeats are encoded precisely by single exons and two of these (EIIIA and EIIIB) are alternatively spliced in a cell type-specific fashion. A third site of alternative splicing (EIIIB) reported here is similar in expression to the previously described EIIIA segment. Both are excluded from mRNA in liver cells and are, therefore, absent from plasma fibronectin. These two alternative splices, plus a third one (V) reported previously, can occur in all possible combinations giving 12 fibronectin mRNAs from a single gene. These splicing variations account for most but not all of the known fibronectin subunit variants. We report investigations designed to detect other regions of alternative splicing. We also show that the pattern of alternative splicing is somewhat altered on oncogenic transformation.     

How prevalent is functional alternative splicing in the human genome?

Comparative analyses of ESTs and cDNAs with genomic DNA predict a high frequency of alternative splicing in human genes. However, there is an ongoing debate as to how many of these predicted splice variants are functional and how many are the result of aberrant splicing (or 'noise'). To address this question, we compared alternatively spliced cassette exons that are conserved between human and mouse with EST-predicted cassette exons that are not conserved in the mouse genome. Presumably, conserved exon-skipping events represent functional alternative splicing. We show that conserved (functional) cassette exons possess unique characteristics in size, repeat content and in their influence on the protein. By contrast, most non-conserved cassette exons do not share these characteristics. We conclude that a significant portion of cassette exons evident in EST databases is not functional, and might result from aberrant rather than regulated splicing.     

Positive selection in alternatively spliced exons of human genes

Alternative splicing is a well-recognized mechanism of accelerated genome evolution. We have studied single-nucleotide polymorphisms and human-chimpanzee divergence in the exons of 6672 alternatively spliced human genes, with the aim of understanding the forces driving the evolution of alternatively spliced sequences. Here, we show that alternatively spliced exons and exon fragments (alternative exons) from minor isoforms experience lower selective pressure at the amino acid level, accompanied by selection against synonymous sequence variation. The results of the McDonald-Kreitman test suggest that alternatively spliced exons, unlike exons constitutively included in the mRNA, are also subject to positive selection, with up to 27% of amino acids fixed by positive selection.     

Evidence for the regulation of alternative splicing via complementary DNA sequence repeats

MOTIVATION: While the mechanism for regulating alternative splicing is poorly understood, secondary structure has been shown to be integral to this process. Due to their propensity for forming complementary hairpin loops and their elevated mutation rates, tandem repeated sequences have the potential to influence splicing regulation. RESULTS: An analysis of human intronic sequences reveals a strong correlation between alternative splicing and the prevalence of mono- through hexanucleotide tandem repeats that may engage in complementary pairing in introns that flank alternatively spliced exons. While only 44% of the 18 173 genes in the Human Alternative Splicing Database are known to be alternatively spliced, they contain 84% of the 694 237 intronic complementary repeat pairs. Significantly, the normalized frequency and distribution of repeat sequences, independent of their potential for pairing, are indistinguishable between alternatively spliced and non-alternatively spliced genes. Thus, the increased prevalence of repeats with pairing potential in alternatively spliced genes is not merely a consequence of more repeats or repeat composition bias. These results suggest that complementary repeats may play a role in the regulation of alternative splicing. CONTACT: harold.garner@utsouthwestern.edu.     

Expression of cDNAs encoding mouse cardiac troponin T isoforms: characterization of a large sample of independent clones

We have isolated 52 mouse cardiac troponin-T-encoding cDNA clones (TnT) by specific antibody screening of a λZAPII expression library. Sequencing data from the large sample of independent cDNAs demonstrated relationships among the expression of four alternatively-spliced exons of the cardiac TnT gene, producing seven classes of cDNAs encoding four protein isoforms differing in two variable regions. In the N-terminal variable region and next to the embryonic-specific exon 4, an alternatively spliced exon 3a was identified in 20% of the adult isoforms. The alternatively spliced exon 12, corresponding to a central variable region between the two functional domains of TnT, was found in approx. 79% of the 52 mouse cardiac TnT cDNAs with a single base mutation completely abolishing the splicing at an internal acceptor site. Three novel alternative splicing acceptor sites in the 5'-untranslated portion of exon 2 have been identified with different frequencies.     

Cis requirements for alternative splicing of the cardiac troponin T pre-mRNA.

The cardiac troponin T (cTNT) pre-mRNA splices 17 exons contiguously but alternatively splices (includes or excludes) the fifth exon. Because both alternative splice products are processed from the same pre-mRNA species, the cTNT pre-mRNA must contain cis-acting sequences which specify exon 5 as an alternative exon. A cTNT minigene (SM-1) transfected into cultured cells produces mRNAs both including and excluding exon 5. The junctions of exons 4-5-6 and 4-6 in the cTNT minigene mRNAs are identical to those of endogenous cTNT mRNAs and no other exons are alternatively spliced. Thus, the SM-1 pre-mRNA is correctly alternatively spliced in transfected cells. To circumscribe the pre-mRNA regions which are required for the alternative nature of exon 5, we have constructed a systematic series of deletion mutants of SM-1. Transfection of this series demonstrates that a 1200 nt pre-mRNA region containing exons 4, 5, and 6 is sufficient to direct alternative splicing of exon 5. Within this region are two relatively large inverted repeats which potentially sequester the alternative exon via intramolecular base-pairing. Such sequestration of an alternative exon is consistent with models which propose pre-mRNA conformation as being determinative for alternative splicing of some pre-mRNAs. However, deletion mutants which remove the majority of each of the inverted repeats retain the ability to alternatively splice exon 5 demonstrating that neither is required for cTNT alternative splice site selection. Taken together, deletion analysis has limited cis elements required for alternative splicing to three small regions of the pre-mRNA containing exons 4, 5, and 6. In addition, the cTNT minigene pre-mRNA expresses both alternative splice products in a wide variety of cultured non-muscle cells as well as in cultured striated muscle cells, although expression of the cTNT pre-mRNA is normally restricted to striated muscle. This indicates that cis elements involved in defining the cTNT exon 5 as an alternative exon do not require muscle-specific factors in trans to function.     

Organization of the Human Protein 4.1 Genomic Locus: New Insights into the Tissue-Specific Alternative Splicing of the Pre-mRNA

Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin–actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones spanning over 200 kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3′-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3′ UTR, over 3 kb, accounts for one-half of the mature mRNA.     

Fibronectin splice variants: understanding their multiple roles in health and disease using engineered mouse models

The extracellular matrix (ECM) is a highly dynamic network of proteins, glycoproteins, and proteoglycans. Numerous diseases result from mutation in genes coding for ECM proteins, but only recently it has been reported that mutations in the fibronectin (FN) gene were associated with a human disorder. FN is one of the main components of the ECM. It generates protein diversity through alternative splicing of a single pre-mRNA, having at least 20 different isoforms in humans. The precise function of these protein isoforms has remained obscure in most cases. Only in the recent few years, it was possible to shed light on the multiple roles of the alternatively spliced FN isoforms. This substantial progress was achieved basically with the knowledge derived from engineered mouse models bearing subtle mutations in specific FN domains. These data, together with a recent report associating mutations in the FN gene to a form of glomerulopathy, clearly show that mutations in constitutive exons or misregulation of alternatively spliced domains of the FN gene may have nonlethal pathological consequences. In this review, we focus on the pathological consequences of mutations in the FN gene, by connecting the function of alternatively spliced isoforms of fibronectin to human diseases.     

Polarized regulation of fibronectin secretion and alternative splicing by transforming growth factor

Fibronectin is a multifunctional protein that is synthesized in several different forms that result from alternative splicing of mRNA. Although expression of splicing variants appears to be both developmentally regulated and tissue-specific, the functional significance of these isoforms is largely unknown. We found that cultured airway epithelial cells vectorially secrete two distinct species of fibronectin, one which contains the alternatively spliced EIIIA region (EIIIA+) and one in which the EIIIA segment is spliced out (EIIIA-). Fibronectin containing the EIIIA region is preferentially secreted apically. Although both apical and basal stimulation with transforming growth factor beta 1 increased fibronectin synthesis, the secretory response differed depending on which surface was being stimulated. Apical secretion of fibronectin and expression of EIIIA+ fibronectin mRNA increased only after apical stimulation. These data demonstrate a novel mechanism for the polarized regulation of targeted secretion and alternative splicing of fibronectin and suggest that the EIIIA segment may act as a targeting signal for the vectorial secretion of fibronectin.     

Genomic organization of the channel catfish CD45 functional gene and CD45 pseudogenes

CD45 is a transmembrane protein tyrosine phosphatase, which in mammals plays an important role in T and B cell receptor and cytokine signaling. Recently, a catfish cDNA was shown to contain all characteristic CD45 features: an alternatively spliced amino-terminus, a cysteine-rich region, three fibronectin domains, a transmembrane region, and two phosphotyrosine phosphatase domains. However, analyses of CD45 cDNAs from various catfish lymphoid cell lines demonstrated that catfish CD45 is unique in that it contains a large number of alternatively spliced exons. Sequence analyses of cDNAs derived from the catfish clonal B cell line 3B11 indicated that this cell line expresses up to 13 alternatively spliced exons. Furthermore, sequence similarity among the alternatively spliced exons suggested duplication events. To establish the exact number and organization of alternatively spliced exons, a bacterial artificial chromosome library was screened, and the catfish functional CD45 gene plus six CD45 pseudogenes were sequenced. The catfish functional CD45 gene spans 37 kb and contains 49 exons. In comparison, the human and pufferfish CD45 genes consist of 34 and 30 exons, respectively. This difference in the otherwise structurally conserved catfish gene is due to the presence of 18 alternatively spliced exons that were likely derived through several duplication events. In addition, duplication events were also likely involved in generating the six pseudogenes, truncated at the 3 ends. A similarly 3 truncated CD45 pseudogene is also present in the pufferfish genome, suggesting that this specific CD45 gene duplication occurred before catfish and pufferfish diverged (400 million years ago).     

Applying genetic programming to the prediction of alternative mRNA splice variants

Genetic programming (GP) can be used to classify a given gene sequence as either constitutively or alternatively spliced. We describe the principles of GP and apply it to a well-defined data set of alternatively spliced genes. A feature matrix of sequence properties, such as nucleotide composition or exon length, was passed to the GP system "Discipulus." To test its performance we concentrated on cassette exons (SCE) and retained introns (SIR). We analyzed 27,519 constitutively spliced and 9641 cassette exons including their neighboring introns; in addition we analyzed 33,316 constitutively spliced introns compared to 2712 retained introns. We find that the classifier yields highly accurate predictions on the SIR data with a sensitivity of 92.1% and a specificity of 79.2%. Prediction accuracies on the SCE data are lower, 47.3% (sensitivity) and 70.9% (specificity), indicating that alternative splicing of introns can be better captured by sequence properties than that of exons.     

A computational and experimental approach toward a priori identification of alternatively spliced exons

Alternative splicing is a powerful means of regulating gene expression and enhancing protein diversity. In fact, the majority of metazoan genes encode pre-mRNAs that are alternatively spliced to produce anywhere from two to tens of thousands of mRNA isoforms. Thus, an important part of determining the complete proteome of an organism is developing a catalog of all mRNA isoforms. Alternatively spliced exons are typically identified by aligning EST clusters to reference mRNAs or genomic DNA. However, this approach is not useful for genomes that lack robust EST coverage, and tools that enable accurate prediction of alternatively spliced exons would be extraordinarily useful. Here, we use comparative genomics to identify, and experimentally verify, potential alternative exons based solely on their high degree of conservation between Drosophila melanogaster and D. pseudoobscura. At least 40% of the exons that fit our prediction criteria are in fact alternatively spliced. Thus, comparative genomics can be used to accurately predict certain classes of alternative exons without relying on EST data.