ASDB: database of alternatively spliced genes

Version 2.1 of ASDB (Alternative Splicing Data Base) contains 1922 protein and 2486 DNA sequences. The protein entries from SWISS-PROT are joined into clusters corresponding to alternatively spliced variants of one gene. The DNA division consists of complete genes with alternative splicing mentioned or annotated in GenBank. The search engine allows one to search over SWISS-PROT and GenBank fields and then follow the links to all variants. The database can be assessed at the URL http://cbcg.nersc.gov/asdb     

EDAS, databases of alternatively spliced human genes

EDAS, a database of alternatively spliced human genes, contains data on the alignment of proteins, mRNAs, and EST. It contains information on all exons and introns observed, as well as elementary alternatives formed from them. The database makes it possible to filter the output data by changing the cut-off threshold by the significance level. The database is accessible at http://www.gene-bee.msu.ru/edas/.     

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.     

Identification of genes alternatively spliced in developing maize endosperm

• The process of alternative splicing is critical for the regulation of growth and development of plants. Thus far, little is known about the role of alternative splicing in the regulation of maize (Zea mays L.) endosperm development.
• RNA sequencing (RNA‐seq) data of endosperms from two maize inbred lines, Mo17 and Ji419, at 15 and 25 days after pollination (DAP), respectively, were used to identify genes that were alternatively spliced during endosperm development. Intron retention (IR) in GRMZM2G005887 was further validated using PCR and re‐sequencing technologies.
• In total, 49,000 alternatively spliced events and ca. 20,000 alternatively spliced genes were identified in the two maize inbred lines. Of these, 30 genes involved in amino acid biosynthesis and starch biosynthesis were identified, with IR occurring only in a specific sample, and were significantly co‐expressed with ten well‐known genes related to maize endosperm development. Moreover, IR in GRMZM2G005887, which encodes a cysteine synthase, was confirmed to occur only in the endosperm of Mo17 at 15 DAP, resulting in the retention of a 121‐bp fragment in its 5′ untranslated region. Two cis‐acting regulatory elements, CAAT‐box and TATA‐box were observed in the retained fragment in Mo17 at 15 DAP; this could regulate the expression of this gene and influence endosperm development.
• The results suggest that the 30 genes with IR identified herein might be associated with maize endosperm development, and are likely to play important roles in the developing maize endosperm.

     

Database and analyses of known alternatively spliced genes in plants

Alternative splicing is an important cellular mechanism that increases the diversity of gene products. The number of alternatively spliced genes reported so far in plants is much smaller than that in mammals, but is increasing as a result of the explosive growth of available EST and genomic sequences. We have searched for all alternatively spliced genes reported in GenBank and PubMed in all plant species under Viridiplantae. After careful merging and manual review of the search results, we obtained a comprehensive, high-quality collection of 168 genes reported to be alternatively spliced in plants, spanning 44 plant species (March 22, 2003 update). We developed a relational database with Web-based user interface to store and present the data, named the Plant Alternative Splicing Database (PASDB), freely available at http://pasdb.genomics.org.cn. We analyzed the functional categories that these genes belong to using the Gene Ontology. We also analyzed in detail the biological roles and gene structures of the four genes that are known to be alternatively spliced in more than one plant species. Finally, we studied the structural features of the splice sites in the alternatively spliced genes.     

Retroviral expression of alternatively spliced forms of rat fibronectin

We describe the construction in retroviral vectors and the expression of recombinant rat fibronectin (FN) cDNAs corresponding with the various alternatively spliced forms of FN. In NIH 3T3 cells, the exogenous rat FN subunits are efficiently secreted as heterodimers with endogenous mouse subunits. In contrast, in lymphoid WEHI231 cells, there is no endogenous FN synthesis and the recombinant FNs are secreted and can be purified as homogeneous proteins. We show that the purified recombinant FNs are biochemically and biologically functional. In basic assays for adhesion, spreading, cytoskeletal organization, and migration using various established adherent cell lines, different forms of FNs containing the different alternatively spliced segments show no marked differences in activity. We have used these recombinant FNs to investigate three systems in which earlier results had suggested potential differences between different forms of FN. First, all forms tested appear equally active in restoring normal morphology to a transformed cell line. Second, we detect minor differences in their ability to assemble into preexisting extracellular matrices. Finally, we report that only those forms of FN that contain the V segment will promote the spreading of a lymphoid cell line indicating that this segment confers additional biological functions for some cell types, a result that confirms and extends earlier data. These homogeneous, biologically active recombinant FNs will allow further studies of the role of the alternatively spliced segments of FN.     

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.     

Tissue-specific expression of two alternatively spliced insulin receptor mRNAs in man

Two previously reported insulin receptor cDNA sequences differ by 36 base pairs (bp) in the distal alpha-subunit, suggesting that alternative mRNA splicing within the coding region may occur (two insulin receptor isoforms). We developed a quantitative modification of the polymerase chain reaction technique in order to detect and characterize differential mRNA splicing at this site within the distal alpha-subunit. Using RNA derived from a variety of human cell types, we detected two polymerase chain reaction-amplified cDNA species reflecting the presence or absence of the above 36 nucleotides. Identity of the two cDNA species was confirmed by Southern blots, the use of a BANI restriction site present only in the 36 base pair segment and dideoxy sequencing. The relative expression of the two mRNA forms varied markedly in a tissue-specific manner. Buffy coat leukocytes and Epstein-Barr virus-transformed lymphocytes express only the shorter mRNA. Placenta expresses both species equally; muscle, isolated adipocytes and cultured fibroblasts express somewhat more of the longer mRNA (relative ratios of mRNA abundance of 1.51, 3.18, and 2.77, respectively); liver expresses mostly the longer mRNA (relative ratio of 9.8). In RNA derived from cultured and fresh cells from patients with several states of insulin resistance, the relative expression of the two mRNA species was similar to results obtained with comparable normal tissues. Although the functional significance of alternative splicing of the insulin receptor mRNA is unknown, differential expression of these two receptor mRNAs may provide a structural basis for previously observed tissue-specific differences in insulin binding and action.     

Characterization and expression of a novel alternatively spliced human angiopoietin-2

Angiopoietin-2 (Ang2) is a naturally occurring antagonist of angiopoietin-1 (Ang1) that competes for binding to the Tie2 receptor and blocks Ang1-induced Tie2 autophosphorylation during vasculogenesis. Using the polymerase chain reaction, we isolated a cDNA encoding a novel shorter form of Ang2 from human umbilical vein endothelial cell cDNA and have designated it angiopoietin-2(443) (Ang2(443)), because it contains 443 amino acids. Part of the coiled-coil domain (amino acids 96-148) is absent in Ang2(443) because of alternative splicing of the gene. Like Ang2, recombinant Ang2(443) expressed in COS-7 cells is secreted as a glycosylated homodimeric protein. Recombinant Ang2(443) binds to the Tie2 receptor but does not induce Tie2 phosphorylation. Pre-occupation of Ang2(443) on Tie2 inhibits Ang1 or Ang2 binding and inhibits Ang1-induced phosphorylation. Expression of Ang2(443) mRNA is detectable in primary endothelial cells, several nonendothelial tumor cell lines, and primary tumor tissues. Interestingly, two cervical carcinoma cell lines express relatively moderate levels of Ang2(443) mRNA and protein. Macrophages express mainly Ang2 mRNA, but the expression of Ang2(443) mRNA is temporarily up-regulated during macrophage differentiation. These results suggest that Ang2(443) is a functional antagonist of Ang1 and could be an important regulator of angiogenesis during some tumorigenic and inflammatory processes.