Recognition of unknown conserved alternatively spliced exons

The split structure of most mammalian protein-coding genes allows for the potential to produce multiple different mRNA and protein isoforms from a single gene locus through the process of alternative splicing (AS). We propose a computational approach called UNCOVER based on a pair hidden Markov model to discover conserved coding exonic sequences subject to AS that have so far gone undetected. Applying UNCOVER to orthologous introns of known human and mouse genes predicts skipped exons or retained introns present in both species, while discriminating them from conserved noncoding sequences. The accuracy of the model is evaluated on a curated set of genes with known conserved AS events. The prediction of skipped exons in the approximately 1% of the human genome represented by the ENCODE regions leads to more than 50 new exon candidates. Five novel predicted AS exons were validated by RT-PCR and sequencing analysis of 15 introns with strong UNCOVER predictions and lacking EST evidence. These results imply that a considerable number of conserved exonic sequences and associated isoforms are still completely missing from the current annotation of known genes. UNCOVER also identifies a small number of candidates for conserved intron retention.     

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.     

The gene for human acidic fibroblast growth factor encodes two upstream exons alternatively spliced to the first coding exon

We have isolated two cDNA clones encoding human acidic fibroblast growth factor (aFGF) which represent the utilization of alternative upstream exons in aFGF mRNA. Isolation and sequence analysis of genomic clones spanning the first coding exon and each of the upstream sequences confirms that the divergent 5' sequences are separate exons, spliced alternatively to the first coding exon 34 nucleotides upstream of the initiator AUG codon. Restriction mapping of the genomic clones provides a minimum size estimate of 45 kilobase pairs for the aFGF locus.     

An alternatively spliced exon links intermediate filaments to adhesions

Anchorage of the contractile actomyosin apparatus to the plasma membrane at discrete sites in muscle and non-muscle cells enables the transmission and conversion of force into work, such as muscle contraction and membrane deformation to regulate cell and tissue shape. Assembly, stabilization and turnover of adhesion sites are complex processes that involve structural components, a variety of signalling and adapter molecules, diverse kinases and phosphatases, and phospholipids. The dynamic turnover of adhesions also requires the frequent interaction with other filament systems of the cytoskeleton, in particular with microtubules. How the delivery and activation of all the required components is co-ordinated, however, remains to be fully understood. In the current issue of Biochemical Journal, Sun et al. provide evidence that a specific exon that is exclusively present in the alpha variant of the type IV intermediate filament protein synemin interacts directly with the focal adhesion protein vinculin in its active state. Interaction of adhesion components with intermediate filaments could serve as a general mechanism to regulate cell- and tissue-specific cytoskeleton-membrane attachment.     

Sam68 regulates a set of alternatively spliced exons during neurogenesis

Sam68 (Src-associated in mitosis, 68 kDa) is a KH domain RNA binding protein implicated in a variety of cellular processes, including alternative pre-mRNA splicing, but its functions are not well understood. Using RNA interference knockdown of Sam68 expression and splicing-sensitive microarrays, we identified a set of alternative exons whose splicing depends on Sam68. Detailed analysis of one newly identified target exon in epsilon sarcoglycan (Sgce) showed that both RNA elements distributed across the adjacent introns and the RNA binding activity of Sam68 are necessary to repress the Sgce exon. Sam68 protein is upregulated upon neuronal differentiation of P19 cells, and many Sam68 RNA targets change in expression and splicing during this process. When Sam68 is knocked down by short hairpin RNAs, many Sam68-dependent splicing changes do not occur and P19 cells fail to differentiate. We also found that the differentiation of primary neuronal progenitor cells from embryonic mouse neocortex is suppressed by Sam68 depletion and promoted by Sam68 overexpression. Thus, Sam68 controls neurogenesis through its effects on a specific set of RNA targets.     

Elements regulating an alternatively spliced exon of the rat fibronectin gene.

We have investigated the regulation of splicing of one of the alternatively spliced exons in the rat fibronectin gene, the EIIIB exon. This 273-nucleotide exon is excluded by some cells and included to various degrees by others. We find that EIIIB is intrinsically poorly spliced and that both its exon sequences and its splice sites contribute to its poor recognition. Therefore, cells which recognize the EIIIB exon must have mechanisms for improving its splicing. Furthermore, in order for EIIB to be regulated, a balance must exist between the EIIIB splice sites and those of its flanking exons. Although the intron upstream of EIIIB does not appear to play a role in the recognition of EIIIB for splicing, the intron downstream contains sequence elements which can promote EIIIB recognition in a cell-type-specific fashion. These elements are located an unusually long distance from the exon that they regulate, more than 518 nucleotides downstream from EIIIB, and may represent a novel mode of exon regulation.     

A sequence compilation and comparison of exons that are alternatively spliced in neurons.

Alternative splicing is an important regulatory mechanism to create protein diversity. In order to elucidate possible regulatory elements common to neuron specific exons, we created and statistically analysed a database of exons that are alternatively spliced in neurons. The splice site comparison of alternatively and constitutively spliced exons reveals that some, but not all alternatively spliced exons have splice sites deviating from the consensus sequence, implying diverse patterns of regulation. The deviation from the consensus is most evident at the -3 position of the 3' splice site and the +4 and -3 position of the 5' splice site. The nucleotide composition of alternatively and constitutively spliced exons is different, with alternatively spliced exons being more AU rich. We performed overlapping k-tuple analysis to identify common motifs. We found that alternatively and constitutively spliced exons differ in the frequency of several trinucleotides that cannot be explained by the amino acid composition and may be important for splicing regulation.     

Specific isolation of 3'-terminal exons of human genes by exon trapping.

Exon trapping is a method to functionally clone expressed sequences from genomic DNA. We have previously developed the vector system pETV-SD2, which contains only a splice donor site (SD) followed by a LacZ gene, allowing trapping of internal exons of human genes by blue-white selection. We now describe the adaptation of the same system for the efficient trapping of 3'-terminal exons, by using different RT-PCR primers in a 3' RACE reaction. The addition of a T7 promoter to the RT-PCR products derived from pETV-SD2 allows their amplification in an isothermic amplification reaction called NASBA (nucleic acid sequence-based amplification reaction) and results in a strong signal from amplified 3' exons in addition to a great reduction of non-specific background. As a test for the system, 3' exon trapping was performed using a cosmid containing the alpha-globin gene cluster on chromosome 16. The 3'-terminal exons of the human alpha 1-, zeta 2-, and theta-globin genes were trapped, as well as a correctly spliced and polyadenylated sequence in the 3' flanking region of the alpha 1-globin gene. This exon appears to belong to a previously unidentified gene within the alpha-globin gene cluster. This 3' exon trapping strategy should facilitate the cloning of genes from large genomic regions.     

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.     

Libraries enriched for alternatively spliced exons reveal splicing patterns in melanocytes and melanomas

It is becoming increasingly clear that alternative splicing enables the complex development and homeostasis of higher organisms. To gain a better understanding of how splicing contributes to regulatory pathways, we have developed an alternative splicing library approach for the identification of alternatively spliced exons and their flanking regions by alternative splicing sequence enriched tags sequencing. Here, we have applied our approach to mouse melan-c melanocyte and B16-F10Y melanoma cell lines, in which 5,401 genes were found to be alternatively spliced. These genes include those encoding important regulatory factors such as cyclin D2, Ilk, MAPK12, MAPK14, RAB4, melastatin 1 and previously unidentified splicing events for 436 genes. Real-time PCR further identified cell line-specific exons for Tmc6, Abi1, Sorbs1, Ndel1 and Snx16. Thus, the ASL approach proved effective in identifying splicing events, which suggest that alternative splicing is important in melanoma development.     

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.