A clean data set of EST-confirmed splice sites from Homo sapiens and standards for clean-up procedures.

A clean data set of verified splice sites from Homo sapiens are reported as well as the standards used for the clean-up procedure. The sites were validated by: (i) standard cleaning procedures such as requiring consistency in the annotation of the gene structural elements, completeness of the coding regions and elimination of redundant sequences; (ii) clustering by decision trees coupled with analysis of ClustalW alignments of the translated protein sequence with homologous proteins from SWISS-PROT; (iii) matching against human EST sequences. The sites are categorised as: (i) donor sites, a set of 619 EST-confirmed donor sites, for which 138 are either the sites or the regions around the sites involved in alternative splice events; (ii) acceptor sites, a set of 623 EST-confirmed acceptor sites, for which 144 are either the sites or the regions around the sites are involved in alternative splice events; (iii) genuine splice sites, a set of 392 splice sites wherein both the donor and acceptor sites had EST confirmation and were not involved in any alternative splicing; (iv) alternative splice sites, a set of 209 splice sites wherein both the donor and acceptor sites had EST confirmation and the sites or the regions around them were involved in alternative splicing. A set of nucleotide regions that can be used to generate a control set of false splice sites that have a high confidence of being non-functional are also reported.     

水稻NBS-LRR基因选择性剪接的全基因组检测及分析

选择性剪接是促进基因组复杂性和蛋白质组多样性的一种主要机制,但是对水稻NBS-LRR序列选择性剪接的全基因组分析却未见报道。通过隐马尔柯夫模型搜索,从TIGR数据库里得到了855条编码NBS-LRR基序的序列。利用这些序列在KOME、TIGR基因索引及UniProt三个数据库中进行同源搜索,获得同源的完整cDNA序列、假设一致性序列和蛋白质序列。再利用Spidey和SIM4程序把完整cDNA序列和假设一致性序列联配到相应的BAC序列上来预测选择性剪接。蛋白质序列和基因组序列之间的联配使用tBLASTn。在这875个NBS-LRR基因中,119个基因具有选择性剪接现象,其中包括71内含子保留,20个外显子跳跃,25个选择性起始,16个选择性终止,12个5′端的选择性剪接和16个3′端选择性剪接。大多数选择性剪接都为两个和多个转录本所支持。可以通过访问http://www.bioinfor.org查询这些数据。进而通过生物信息学分析剪接边界发现外显子跳跃和内含子保留的‘GT…AG’的规则不如组成型的保守。这暗示了它们是通过不同的调控机制来指导剪接变构体的形成。通过分析内含子保留对蛋白质的影响,发现选择性剪接的蛋白更倾向于改变其C端氨基酸序列。最后对选择性剪接的组织分布和蛋白质定位进行分析,结果表明选择性剪接的最大类的组织分布是根和愈伤组织。超过1/3剪接变构体的蛋白质定位是质膜和细胞质。这些选择性剪接蛋白可能在抗病信号转导中起到重要作用。     

ASAP: the Alternative Splicing Annotation Project

Recently, genomics analyses have demonstrated that alternative splicing is widespread in mammalian genomes (30-60% of genes reported to have multiple isoforms), and may be one of their most important mechanisms of functional regulation. However, by comparison with other genomics data such as genome annotation, SNPs, or gene expression, there exists relatively little database infrastructure for the study of alternative splicing. We have constructed an online database ASAP (the Alternative Splicing Annotation Project) for biologists to access and mine the enormous wealth of alternative splicing information coming from genomics and proteomics. ASAP is based on genome-wide analyses of alternative splicing in human (30 793 alternative splice relationships found) from detailed alignment of expressed sequences onto the genomic sequence. ASAP provides precise gene exon-intron structure, alternative splicing, tissue specificity of alternative splice forms, and protein isoform sequences resulting from alternative splicing. Moreover, it can help biologists design probe sequences for distinguishing specific mRNA isoforms. ASAP is intended to be a community resource for collaborative annotation of alternative splice forms, their regulation, and biological functions. The URL for ASAP is http://www.bioinformatics.ucla.edu/ASAP.     

Computational comparative analyses of alternative splicing regulation using full-length cDNA of various eukaryotes

We previously reported a computational approach to infer alternative splicing patterns from Mus musculus full-length cDNA clones and microarray data. Although we predicted a large number of unreported splice variants, the general mechanisms regulating alternative splicing were yet unknown. In the present study, we compared alternative exons and constitutive exons in terms of splice-site strength and frequency of potential regulatory sequences. These regulatory features were further compared among five different species: Homo sapiens, M. musculus, Arabidopsis thaliana, Oryza sativa, and Drosophila melanogaster. Solid statistical validations of our comparative analyses indicated that alternative exons have (1) weaker splice sites and (2) more potential regulatory sequences than constitutive exons. Based on our observations, we propose a combinatorial model of alternative splicing mechanisms, which suggests that alternative exons contain weak splice sites regulated alternatively by potential regulatory sequences on the exons.     

Genomic splice-site analysis reveals frequent alternative splicing close to the dominant splice site

Alternative pre-mRNA splicing may be the most efficient and widespread mechanism to generate multiple protein isoforms from single genes. Here, we describe the genomic analysis of one of the most frequent types of alternative pre-mRNA splicing, alternative 5'- and 3'-splice-site selection. Using an EST-based alternative splicing database recording >47,000 alternative splicing events, we determined the frequency and location of alternative 5'- and 3'-splice sites within the human genome. The most common alternative splice sites used in the human genome are located within 6 nucleotides (nt) of the dominant splice site. We show that the EST database overrepresents alternative splicing events that maintain the reading frame, thus supporting the concept that RNA quality-control steps ensure that mRNAs that encode for potentially harmful protein products are destroyed and do not serve as templates for translation. The most frequent location for alternative 5'-splice sites is 4 nt upstream or downstream from the dominant splice site. Sequence analysis suggests that this preference is a consequence of the U1 snRNP binding sequence at the 5'-splice site, which frequently contains a GU dinucleotide 4 nt downstream from the dominant splice site. Surprisingly, approximately 50% of duplicated 3'-YAG splice junctions are subject to alternative splicing. This high probability of alternative 3'-splice-site activation in close proximity of the dominant 3'-splice site suggests that the second step of the splicing may be prone to violate splicing fidelity.     

Alternative splice variants encoding unstable protein domains exist in the human brain

Alternative splicing has been recognized as a major mechanism by which protein diversity is increased without significantly increasing genome size in animals and has crucial medical implications, as many alternative splice variants are known to cause diseases. Despite the importance of knowing what structural changes alternative splicing introduces to the encoded proteins for the consideration of its significance, the problem has not been adequately explored. Therefore, we systematically examined the structures of the proteins encoded by the alternative splice variants in the HUGE protein database derived from long (>4 kb) human brain cDNAs. Limiting our analyses to reliable alternative splice junctions, we found alternative splice junctions to have a slight tendency to avoid the interior of SCOP domains and a strong statistically significant tendency to coincide with SCOP domain boundaries. These findings reflect the occurrence of some alternative splicing events that utilize protein structural units as a cassette. However, 50 cases were identified in which SCOP domains are disrupted in the middle by alternative splicing. In six of the cases, insertions are introduced at the molecular surface, presumably affecting protein functions, while in 11 of the cases alternatively spliced variants were found to encode pairs of stable and unstable proteins. The mRNAs encoding such unstable proteins are much less abundant than those encoding stable proteins and tend not to have corresponding mRNAs in non-primate species. We propose that most unstable proteins encoded by alternative splice variants lack normal functions and are an evolutionary dead-end.     

AVATAR: a database for genome-wide alternative splicing event detection using large scale ESTs and mRNAs

In the past years, identification of alternative splicing (AS) variants has been gaining momentum. We developed AVATAR, a database for documenting AS using 5,469,433 human EST sequences and 26,159 human mRNA sequences. AVATAR contains 12000 alternative splicing sites identified by mapping ESTs and mRNAs with the whole human genome sequence. AVATAR also contains AS information for 6 eukaryotes. We mapped EST alignment information into a graph model where exons and introns are represented with vertices and edges, respectively. AVATAR can be queried using, (1) gene names, (2) number of identified AS events in a gene, (3) minimal number of ESTs supporting a splicing site, etc. as search parameters. The system provides visualized AS information for queried genes.

Availability     

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.     

Identification of alternative 5'/3' splice sites based on the mechanism of splice site competition

Alternative splicing plays an important role in regulating gene expression. Currently, most efficient methods use expressed sequence tags or microarray analysis for large-scale detection of alternative splicing. However, it is difficult to detect all alternative splice events with them because of their inherent limitations. Previous computational methods for alternative splicing prediction could only predict particular kinds of alternative splice events. Thus, it would be highly desirable to predict alternative 5'/3' splice sites with various splicing levels using genomic sequences alone. Here, we introduce the competition mechanism of splice sites selection into alternative splice site prediction. This approach allows us to predict not only rarely used but also frequently used alternative splice sites. On a dataset extracted from the AltSplice database, our method correctly classified approximately 70% of the splice sites into alternative and constitutive, as well as approximately 80% of the locations of real competitors for alternative splice sites. It outperforms a method which only considers features extracted from the splice sites themselves. Furthermore, this approach can also predict the changes in activation level arising from mutations in flanking cryptic splice sites of a given splice site. Our approach might be useful for studying alternative splicing in both computational and molecular biology.     

An intronic signal for alternative splicing in the human genome

An important level at which the expression of programmed cell death (PCD) genes is regulated is alternative splicing. Our previous work identified an intronic splicing regulatory element in caspase-2 (casp-2) gene. This 100-nucleotide intronic element, In100, consists of an upstream region containing a decoy 3' splice site and a downstream region containing binding sites for splicing repressor PTB. Based on the signal of In100 element in casp-2, we have detected the In100-like sequences as a family of sequence elements associated with alternative splicing in the human genome by using computational and experimental approaches. A survey of human genome reveals the presence of more than four thousand In100-like elements in 2757 genes. These In100-like elements tend to locate more frequent in intronic regions than exonic regions. EST analyses indicate that the presence of In100-like elements correlates with the skipping of their immediate upstream exons, with 526 genes showing exon skipping in such a manner. In addition, In100-like elements are found in several human caspase genes near exons encoding the caspase active domain. RT-PCR experiments show that these caspase genes indeed undergo alternative splicing in a pattern predicted to affect their functional activity. Together, these results suggest that the In100-like elements represent a family of intronic signals for alternative splicing in the human genome.     

Investigation of Tissue-Specific Human Orthologous Alternative Splice Events in Pig

Alternative splicing of pre-mRNA can contribute to differences between tissues or cells either by regulating gene expression or creating proteins with various functions encoded by one gene. The number of investigated alternative splice events in pig has so far been limited. In this study we have investigated alternative splice events detected in humans, in orthologous pig genes. A total of 17 genes with predicted exon skipping events were selected for further studies. The splice events for the selected genes were experimentally verified using real-time quantitative PCR analysis (qPCR) with splice-specific primers in 19 different tissues. The same splice variants as reported in humans were detected in 15 orthologous pig genes, however, the expression pattern predicted in the in silico analyses was only experimentally verified in a few cases. The results support the findings that splice events resulting in preservation of open reading frame are indicative of a functional significance of the splice variants of the gene.     

Investigation of tissue-specific human orthologous alternative splice events in pig

Alternative splicing of pre-mRNA can contribute to differences between tissues or cells either by regulating gene expression or creating proteins with various functions encoded by one gene. The number of investigated alternative splice events in pig has so far been limited. In this study we have investigated alternative splice events detected in humans, in orthologous pig genes. A total of 17 genes with predicted exon skipping events were selected for further studies. The splice events for the selected genes were experimentally verified using real-time quantitative PCR analysis (qPCR) with splice-specific primers in 19 different tissues. The same splice variants as reported in humans were detected in 15 orthologous pig genes, however, the expression pattern predicted in the in silico analyses was only experimentally verified in a few cases. The results support the findings that splice events resulting in preservation of open reading frame are indicative of a functional significance of the splice variants of the gene.     

ProSplicer: a database of putative alternative splicing information derived from protein,mRNA and expressed sequence tag sequence data

ProSplicer is a database of putative alternative splicing information derived from the alignment of proteins, mRNA sequences and expressed sequence tags (ESTs) against human genomic DNA sequences. Proteins, mRNA and ESTs provide valuable evidence that can reveal splice variants of genes. The alternative splicing information in the database can help users investigate the alternative splicing and tissue-specific expression of genes.