A procedure for identifying homologous alternative splicing events

Background  

The study of the functional role of alternative splice isoforms of a gene is a very active area of research in biology. The difficulty of the experimental approach (in particular, in its high-throughput version) leaves ample room for the development of bioinformatics tools that can provide a useful first picture of the problem. Among the possible approaches, one of the simplest is to follow classical protein function annotation protocols and annotate target alternative splice events with the information available from conserved events in other species. However, the application of this protocol requires a procedure capable of recognising such events. Here we present a simple but accurate method developed for this purpose.     

The splice of life: alternative splicing and neurological disease

Splicing of pre-messenger RNA is regulated differently in the brain compared with other tissues. Recognition of aberrations in splicing events that are associated with neurological disease has contributed to our understanding of disease pathogenesis in some cases. Neuron-specific proteins involved in RNA splicing and metabolism are also affected in several neurological disorders. These findings have begun to bridge what we know about the mechanisms regulating neuron-specific splicing and our understanding of neural function and disease.     

Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons

Thousands of tandem alternative splice sites (TASS) give rise to mRNA insertion/deletion variants with small size differences. Recent work has concentrated on the question of biological relevance in general, and the physiological regulation of TASS in particular. We have quantitatively studied 11 representative TASS cases in comparison to one mutually exclusive exon case and two cassette exons (CEs) using a panel of human and mouse tissues, as well as cultured cell lines. Tissues show small but significant differences in TASS isoform ratios, with a variance 4- to 20-fold lower than seen for CEs. Remarkably, in cultured cells, all studied alternative splicing (AS) cases showed a cell-density-dependent shift of isoform ratios with similar time series profiles. A respective genome-wide co-regulation of TASS splicing was shown by next-generation mRNA sequencing data. Moreover, data from human and mouse organs indicate that this co-regulation of TASS occurs in vivo, with brain showing the strongest difference to other organs. Together, the results indicate a physiological AS regulation mechanism that functions almost independently from the splice site context and sequence.     

Cross-species EST alignments reveal novel and conserved alternative splicing events in legumes

Background  

Although originally thought to be less frequent in plants than in animals, alternative splicing (AS) is now known to be widespread in plants. Here we report the characteristics of AS in legumes, one of the largest and most important plant families, based on EST alignments to the genome sequences of Medicago truncatula (Mt) and Lotus japonicus (Lj).     

Discovery of novel splice forms and functional analysis of cancer-specific alternative splicing in human expressed sequences

We report here a genome-wide analysis of alternative splicing in 2 million human expressed sequence tags (ESTs), to identify splice forms that are up-regulated in tumors relative to normal tissues. We found strong evidence (P < 0.01) of cancer-specific splice variants in 316 human genes. In total, 78% of the cancer-specific splice forms we detected are confirmed by human-curated mRNA sequences, indicating that our results are not due to random mis-splicing in tumors; 73% of the genes showed the same cancer-specific splicing changes in tissue-matched tumor versus normal datasets, indicating that the vast majority of these changes are associated with tumorigenesis, not tissue specificity. We have confirmed our EST results in an independent set of experimental data provided by human-curated mRNAs (P-value 10^–5.7). Moreover, the majority of the genes we detected have functions associated with cancer (P-value 0.0007), suggesting that their altered splicing may play a functional role in cancer. Analysis of the types of cancer-specific splicing shifts suggests that many of these shifts act by disrupting a tumor suppressor function. Sur prisingly, our data show that for a large number (190 in this study) of cancer-associated genes cloned originally from tumors, there exists a previously uncharacterized splice form of the gene that appears to be predominant in normal tissue.     

A novel alternative splicing form of excitatory amino acid transporter 1 is a negative regulator of glutamate uptake

Abstract EAAT1 is a major glutamate transporter in the CNS and is required for normal neurotransmission and neuroprotection from excitotoxicity. In the present study, we have identified a novel form of the human EAAT1, named here as EAAT1ex9skip, which lacks the entire exon 9. Quantitative PCR analysis indicates that this variant is expressed throughout the CNS, both in grey matter and axonal tracts, at levels ranging between 10% and 20% of the full-length EAAT1 form. When expressed in HEK293 cells, EAAT1ex9skip mRNA is translated into a truncated protein localized in the endoplasmic reticulum. EAAT1ex9skip has no functional glutamate uptake activity but instead, exerts a dominant negative effect over full-length EAAT1 function. In turn, co-expression of full-length EAAT1 and EAAT1ex9skip variants reduces the insertion of the former into the plasma membrane. Together, these results indicate that the EAAT1ex9skip splice variant is a negative regulator of full-length EAAT1 function in the human brain.     

Identification of a novel alternative splicing of human FGF receptor 4: soluble-form splice variant expressed in human gastrointestinal epithelial cells

Among four closely related members of the FGF receptor family, FGFR 1, 2, and 3 have alternative splicing forms encoded by different exons for the C-terminal half of the third Ig-like domain, but FGFR 4 has no such alternative exon. Furthermore, FGFR 1, 2, and 3 have another splice variant of nontransmembrane type; however, such a variant has not been reported for FGFR 4. While searching for a novel receptor-type tyrosine kinase by RT-PCR, we identified a non-transmembrane-type receptor of FGFR 4 in human intestinal epithelial cell lines (Intestine 407 and Caco-2). Sequence analysis of this receptor revealed that exon 9 coding the single transmembrane domain was displaced by intron 9. Consequently, this variant form was 120 bp shorter than the normal form and had no transmembrane portion. Moreover, the signal sequence in exon 2 was maintained, suggesting that this splice variant is a soluble receptor. This soluble receptor was detected in human gastrointestinal epithelial cells and pancreas, and also in gastric, colon, and pancreatic cancer cell lines. Single cell RT-PCR showed that this soluble receptor was expressed simultaneously with the transmembrane-type receptor in the same cell. Western blot analysis revealed that this receptor was secreted from the transfected COS7 cells. Thus, a soluble-form splice variant of FGFR 4 was identified in human gastrointestinal epithelial cells and cancer cells. This is the first report of alternative splicing of FGFR 4.     

EuSplice: a unified resource for the analysis of splice signals and alternative splicing in eukaryotic genes

MOTIVATION: Despite increased availability of genome annotation data, a comprehensive resource for in-depth analysis of splice signal distributions and alternative splicing (AS) patterns in eukaryote genomes is still lacking. To meet this need, we have developed EuSplice--a unique splice-centric database which provides reliable splice signal and AS information for 23 eukaryotes. RESULTS: The EuSplice database contains 95,822 AS events and 2.1 million splice signals associated with over 270,000 protein-coding genes. The intuitive, user-friendly EuSplice web interface has powerful data mining and graphics capabilities for inter-genomic comparative analysis of splice signals, putative cryptic splice sites and AS events. Moreover, the seamless integration of splicing data to extensive gene-specific annotations, such as homolog annotations, functional information, mutations and sequence details makes EuSplice a powerful one-stop information resource for investigating the molecular mechanisms of complex splicing events, disease associations and the evolution of splicing in eukaryotes. AVAILABILITY: http://66.170.16.154/EuSplice. SUPPLEMENTARY INFORMATION: Supplementary tables and figures at Bioinfo online.     

Violating the splicing rules: TG dinucleotides function as alternative 3' splice sites in U2-dependent introns

Background  

Despite some degeneracy of sequence signals that govern splicing of eukaryotic pre-mRNAs, it is an accepted rule that U2-dependent introns exhibit the 3' terminal dinucleotide AG. Intrigued by anecdotal evidence for functional non-AG 3' splice sites, we carried out a human genome-wide screen.     

真菌基因可变剪接可视化分析的新方法:ZOOM软件的应用扩展（英文）

可变剪接是引起蛋白多样性的主要机制之一,而转录组reads的重新定位是获取可变剪接位点的有效方法,适合在基因组较小的真菌中应用。ZOOM软件是一款可在window系统下运行的reads可视化定位软件,被广泛用于下一代基因组测序(NGS)的reads定位及单碱基多态性位点(SNP)的发掘。本文发现该软件分析可变剪接的新用途,并以禾谷镰刀菌Fusarium graminearum的4个基因为例详细描述该方法在真菌可变剪接位点识别中的应用,这些结果均获得RT-PCR的验证。