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PubMed2003

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.

Alternative Splicing↗

PubMed2004

Alternative splicing and genomic stability.

Alternative splicing allows an organism to make different proteins in different cells at different times, all from the same gene. In a cell that uses alternative splicing, the total length of all the exons is much shorter than in a cell that encodes the same set of proteins without alternative splicing. This economical use of exons makes genes more stable during reproduction and development because a genome with a shorter exon length is more resistant to harmful mutations. Genomic stability may be the reason why higher vertebrates splice alternatively. For a broad class of alternatively spliced genes, a formula is given for the increase in their stability.

Alternative Splicing↗

PubMed2000

Alternative splicing of protein 4.1R exon 16: ordered excision of flanking introns ensures proper splice site choice.

Alternative splicing plays a major role in regulating tissue-specific expression of cytoskeletal protein 4.1R isoforms. In particular, expression of the protein's functionally critical spectrin-actin binding domain, essential for maintenance of red cell membrane mechanical properties, is governed by a developmentally regulated splicing switch involving alternative exon 16. Using a model 3-exon 4.1R pre-messenger RNA (pre-mRNA), we explored the sequence requirements for excision of the introns flanking exon 16. These studies revealed that splicing of this alternative exon occurs preferentially in an ordered fashion. The first step is excision of the downstream intron to join exons 16 and 17, followed by excision of the upstream intron. Constructs designed to test the converse pathway were spliced less efficiently and with less fidelity, in part due to activation of a cryptic 5' splice site in exon 16. This downstream-first model for ordered splicing is consistent with the hypothesis that regulated alternative splicing requires cooperation between multiple exonic and/or intronic regulatory elements whose spatial organization is critical for recruitment of appropriate splicing factors. Our results predict that exon 16 splicing is regulated at the first step-excision of the downstream intron-and that cells unable to catalyze this step will exhibit exon 16 skipping. In cells that include exon 16, adherence to an ordered pathway is important for efficient and accurate production of mature 4.1R mRNA encoding an intact spectrin-actin binding domain. (Blood. 2000;95:692-699)

Alternative Splicing↗

PubMed1995

Titration of serine/arginine (SR) splicing factors during adenoviral infection modulates E1A pre-mRNA alternative splicing.

Alternative splicing of the adenovirus-2 E1A pre-mRNA involves the use of three 5' splice sites and is modulated during infection because the 13S mRNA and 9S mRNA reactions are predominant during the early and late periods, respectively. We had previously reproduced in vitro the 13S to 9S modulation with nuclear extracts isolated from infected HeLa cells and shown that high molecular weight viral RNAs are involved in this modulation, most likely by sequestering or titrating general splicing factors. To further test this hypothesis, we titrated splicing factors from an uninfected nuclear extract using competitor RNA or by progressive inactivation of splicing factors with monoclonal antibodies. We found that the 13S to 9S modulation occurs when titrating only with certain RNAs (essentially adenoviral RNAs), and also by progressively inactivating the 9G8 SR splicing factor. The demonstration that late nuclear extracts contain levels of active SR splicing factors limiting for the 13S reaction has been made by complementation experiments. We show that late nuclear extracts do not complement SR factor-deficient extracts, whereas late extracts treated with micrococcal nuclease complement them. Furthermore, complementation of late nuclear extracts with each of the three 30-35-kDa SR factors (9G8, SC35, and SF2/ASF) restores an efficient 13S mRNA reaction. Thus, our results provide evidence that the 13S to 9S modulation is triggered through a titration of SR factors required for the 13S mRNA reaction by major late transcripts that accumulate in nuclei late in infection.

Adenoviridae↗

PubMed2006

Fast rate of evolution in alternatively spliced coding regions of mammalian genes.

BACKGROUND: At least half of mammalian genes are alternatively spliced. Alternative isoforms are often genome-specific and it has been suggested that alternative splicing is one of the major mechanisms for generating protein diversity in the course of evolution. Another way of looking at alternative splicing is to consider sequence evolution of constitutive and alternative regions of protein-coding genes. Indeed, it turns out that constitutive and alternative regions evolve in different ways. RESULTS: A set of 3029 orthologous pairs of human and mouse alternatively spliced genes was considered. The rate of nonsynonymous substitutions (dN), the rate of synonymous substitutions (dS), and their ratio (omega = dN/dS) appear to be significantly higher in alternatively spliced coding regions compared to constitutive regions. When N-terminal, internal and C-terminal alternatives are analysed separately, C-terminal alternatives appear to make the main contribution to the observed difference. The effects become even more pronounced in a subset of fast evolving genes. CONCLUSION: These results provide evidence of weaker purifying selection and/or stronger positive selection in alternative regions and thus one more confirmation of accelerated evolution in alternative regions. This study corroborates the theory that alternative splicing serves as a testing ground for molecular evolution.

Alternative Splicing↗

PubMed2005

Neuronal proteins custom designed by alternative splicing.

The evolution of alternative splicing in eukaryotes greatly expanded the number of functionally distinct proteins that could be produced from a finite gene pool. Extensive in the brains of higher organisms, alternative splicing might be the primary mechanism for generating the spectrum of protein activities that support complex brain functions. Alternative splicing is controlled at the level of individual neurons to custom design proteins for optimal performance. The expression profiles of splice isoforms are modified during development and as neuronal activity changes. Alternative splicing can lead to incremental, long lasting changes in ion channel and receptor activities, independent of changes in gene transcription. Recent studies of tissue-specific splicing factors are revealing how coordinated alterations in alternative splicing of RNA transcripts control synaptic function.

Alternative Splicing↗

PubMed2001

Model system for evaluation of alternative splicing: exon skipping.

Alternative splicing of the primary RNA transcript is a common mechanism for generating protein diversity. A model system was developed to study this process in vitro that is useful for evaluation of splicing of transcripts expressed in cells that do not grow well in culture. The system was used to analyze skipping of exon 4 of the amelogenin message, normally expressed in ameloblast cells for a short interval during tooth enamel development. Amelogenins are highly conserved proteins resulting from extensive alternative splicing, with domains involved in a range of functions, including mineral formation and intercellular signaling. In the bovine gene, the very short intron 4 was predicted to inhibit inclusion of exon 4, because in murine ameloblasts, exon 4 is detectably included in mRNA, and intron 4 is longer than the bovine counterpart. Bovine intron 4 was lengthened, and this size increase enhanced exon 4 inclusion sixfold to eightfold, although splice site selection was inaccurate. Intron length, therefore, is not the sole determinant controlling amelogenin exon 4 inclusion, and cis-acting inhibitory elements may also be involved in exon skipping. This vector system allows evaluation of splicing of a tissue-specific RNA by focusing on exons of interest through transfection of heterologous cultured cells without complications attributable to background transcription of the gene being evaluated.

Alternative Splicing↗

PubMed2003

Alternative splicing of transcripts from crtI and crtYB genes of Xanthophyllomyces dendrorhous.

Xanthophyllomyces dendrorhous is one of the relevant sources of the carotenoid astaxanthin. In this paper, we describe for the first time cloning of unexpected cDNAs obtained from the crtI and crtYB genes of X. dendrorhous strain UCD 67-385. The cDNA of the crtI gene conserves 80 bp of the first intron, while the cDNA of the crtYB gene conserves 55 bp of the first intron and lacks 111 bp of the second exon. The crtI and crtYB RNAs could be spliced in alternative splice sites, which produced alternative transcripts which could not be translated to active CRTI and CRTYB proteins since they had numerous stop codons in their sequences. The ratio of mature mRNA to alternative mRNA for the crtI gene decreased as a function of the age of the culture, while the cellular content of carotenoids increased. It is possible that splicing to mature or alternative transcripts could regulate the cellular concentrations of phytoene desaturase and phytoene synthase-lycopene cyclase proteins, depending on the physiological or environmental conditions.

Alkyl and Aryl Transferases↗

PubMed1995

Adhesive interactions between alternatively spliced CD44 isoforms.

Alternative splicing of a series of 10 contiguous exons present within the CD44 gene can generate a large number of differentially expressed CD44 isoforms that contain additional peptide sequences of varying length inserted into a single site within the extracellular domain of the molecule proximal to the membrane spanning domain. Although distinct functions have been ascribed to certain of these isoforms, the effect of particular inserted domains on the ligand-binding specificity of the CD44 molecule remains unclear. In the present study, we demonstrate that while CD44H, the major CD44 isoform expressed on resting hemopoietic cells, and CD44R1, an alternatively spliced isoform present on transformed epithelial cells and certain activated and/or malignant hemopoietic cell types, can both bind avidly to hyaluronan, only CD44R1 can promote homotypic cellular aggregation when expressed in the CD44-negative murine lymphoma cell line TIL1. Experiments in which TIL1 cells transduced with different CD44 isoforms were tested for their ability to adhere to one another or to COS7 cells transfected with CD44R1, indicated that CD44R1 can recognize and bind a common determinant present on both CD44H and CD44R1. Monoclonal antibody blocking studies suggest further, that the determinant recognized by CD44R1 is located in a region of the CD44 molecule distinct from that involved in hyaluronan binding.

Alternative Splicing↗

PubMed1998

Splicing of 5' introns dictates alternative splice selection of acetylcholinesterase pre-mRNA and specific expression during myogenesis.

Splicing of alternative exon 6 to invariant exons 2, 3, and 4 in acetylcholinesterase (AChE) pre-mRNA results in expression of the prevailing enzyme species in the nervous system and at the neuromuscular junction of skeletal muscle. The structural determinants controlling splice selection are examined in differentiating C2-C12 muscle cells by selective intron deletion from and site-directed mutagenesis in the Ache gene. Transfection of a plasmid lacking two invariant introns (introns II and III) within the open reading frame of the Ache gene, located 5' of the alternative splice region, resulted in alternatively spliced mRNAs encoding enzyme forms not found endogenously in myotubes. Retention of either intron II or III is sufficient to control the tissue-specific pre-mRNA splicing pattern prevalent in situ. Further deletions and branch point mutations revealed that upstream splicing, but not the secondary structure of AChE pre-mRNA, is the determining factor in the splice selection. In addition, deletion of the alternative intron between the splice donor site and alternative acceptor sites resulted in aberrant upstream splicing. Thus, selective splicing of AChE pre-mRNA during myogenesis occurs in an ordered recognition sequence in which the alternative intron influences the fidelity of correct upstream splicing, which, in turn, determines the downstream splice selection of alternative exons.

Acetylcholinesterase↗

PubMed1990

A novel pathway for alternative splicing: identification of an RNA intermediate that generates an alternative 5' splice donor site not present in the primary transcript of AMPD1.

AMP deaminase (AMPD) is a central enzyme in eucaryotic energy metabolism, and tissue-specific as well as stage-specific isoforms are found in many vertebrates. This study demonstrates the AMPD1 gene product in rat is alternatively spliced. The second exon, a 12-base miniexon, was found to be excluded or included in a tissue-specific and stage-specific pattern. This example of cassette splicing utilizes a unique pathway through an RNA intermediate that generates an alternative 5' splice donor site at the point where exon 2 is ligated to exon 1. In the analogous intermediate of human AMPD1, the potential 5' splice donor site created at the boundary of exon 1 and exon 2 was a poor substrate for splicing because of differences in exon 2 sequences, and human AMPD1 was not alternatively spliced. These results demonstrate that in some cases alternative splicing may proceed through an RNA intermediate that generates an alternative splice donor site not present in the primary transcript. Discrimination between alternative 5' splice donor sites in the RNA intermediate of AMPD1 is apparently controlled by tissue-specific and stage-specific signals.

AMP Deaminase↗

PubMed1999

Frequent alternative splicing of human genes.

Alternative splicing can produce variant proteins and expression patterns as different as the products of different genes, yet the prevalence of alternative splicing has not been quantified. Here the spliced alignment algorithm was used to make a first inventory of exon-intron structures of known human genes using EST contigs from the TIGR Human Gene Index. The results on any one gene may be incomplete and will require verification, yet the overall trends are significant. Evidence of alternative splicing was shown in 35% of genes and the majority of splicing events occurred in 5' untranslated regions, suggesting wide occurrence of alternative regulation. Most of the alternative splices of coding regions generated additional protein domains rather than alternating domains.

5' Untranslated Regions↗

PubMed2006

Identification of candidates for tumor-specific alternative splicing in the thyroid.

Alternative splicing is the differential processing of exon junctions to produce a new transcript variant from one gene. Some aberrant splicing, however, has been shown to be cancer specific. Identification of these specific splice variations will provide important insight into the molecular mechanism of normal cellular physiology as well as the disease processes. To gain knowledge about whether alternative splicing is linked to thyroid tumorigenesis, we used our prediction database to select targets for analysis. Fifteen putatively new alternative splicing isoforms were selected on the basis of their expression in thyroid libraries and/or their origin in genes previously associated with carcinogenesis. Using a set of 66 normal, benign, and malignant thyroid tissue samples, new splicing events were confirmed by RT-PCR for 13 of 15 genes (a validation rate of 87%). In addition, new alternative splicing isoforms not predicted by the system and not previously described in public databases were identified. Five genes (PTPN18, ABI3BP, PFDN5, SULF2, and ST5) presented new and/or additional unpredicted isoforms differentially expressed between malignant and benign or normal thyroid tissues, confirmed by sequencing. PTPN18, ABI3BP, and PFDN5 revealed a statistically significant differential splicing profile. In addition, real-time PCR analysis revealed that expression of an alternative PFDN5 variant was higher in malignant lesions than in benign lesions or normal tissues.

Alternative Splicing↗

PubMed1997

Splice-junction elements and intronic sequences regulate alternative splicing of the Drosophila myosin heavy chain gene transcript.

The Drosophila muscle myosin heavy chain (Mhc) gene primary transcript contains five alternatively spliced exon groups (exon 3, 7, 9, 11 and 15), each of which contains two to five mutually exclusive members. Individual muscles typically select a specific alternative exon from each group for incorporation into the processed message. We report here on the cis-regulatory mechanisms that direct the processing of alternative exons in Mhc exon 11 in individual muscles using transgenic reporter constructs, RT-PCR and directed mutagenesis. The 6.0-kilobase exon 11 domain is sufficient to direct the correct processing of exon 11 alternatives, demonstrating that the alternative splicing cis-regulatory elements are local to Mhc exon 11. Mutational analysis of Mhc exon 11 reveals that the alternative exon nonconsensus 5'-splice donors are essential for alternative splicing regulation in general, but do not specify alternative exons for inclusion in individual muscles. Rather, we show, through exon substitutions and deletion analyses, that a 360-nucleotide intronic domain precisely directs the normal processing of one exon, Mhc exon 11e, in the indirect flight muscle. These and other data indicate that alternative exons are regulated in appropriate muscles through interactions between intronic alternative splice-specificity elements, nonconsensus exon 11 splice donors and, likely, novel exon-specific alternative splicing factors.

Alternative Splicing↗

PubMed1999

Circular dystrophin RNAs consisting of exons that were skipped by alternative splicing.

Exon skipping by alternative splicing and circular RNA formation are proposed to be interrelated events. Since multiple patterns of alternative splicing have been demonstrated in both the 5' and 3' regions of the dystrophin gene, the dystrophin transcript in skeletal muscle cells provides a model system in which this idea is tested. Nine circular RNAs that were expected to result from known exon skipping patterns in the 5' region of this gene were in fact identified, but three other circular RNAs expected to result from other known exon skipping reactions in this region could not be detected. The identification of two unexpected circular RNAs led to the discovery of two novel alternative splicing reactions. One circular RNA originating in the 3' region of the gene was identified but it lacked one small sized exon compared with the expected exon structure. Circular RNAs from the 5' region of the dystrophin gene could not be detected in Duchenne muscular dystrophy patients who have deletions of one or more exons in this segment of the gene, even though alternative splicing products were identified. These results showed that circular RNA formation is not necessarily linked to exon skipping and suggest that an undetermined factor regulates circular RNA formation.

Alternative Splicing↗

PubMed2001

Alternative spliced transcripts as cancer markers.

Eukaryotic mRNAs are transcribed as precursors containing their intronic sequences. These are subsequently excised and the exons are spliced together to form mature mRNAs. This process can lead to transcript diversification through the phenomenon of alternative splicing. Alternative splicing can take the form of one or more skipped exons, variable position of intron splicing or intron retention. The effect of alternative splicing in expanding protein repertoire might partially underlie the apparent discrepancy between gene number and the complexity of higher eukaryotes. It is likely that more than 50% form. Many cancer-associated genes, such as CD44 and WT1 are alternatively spliced. Variation of the splicing process occurs during tumor progression and may play a major role in tumorigenesis. Furthermore, alternatively spliced transcripts may be extremely useful as cancer markers, since it appears likely that there may be striking contrasts in usage of alternatively spliced transcript variants between normal and tumor tissue than in alterations in the general levels of gene expression.

Alternative Splicing↗

PubMed2005

Detection of alternatively spliced transcripts in leukemia cell lines by minisequencing on microarrays.

BACKGROUND: Recent genome-wide expression studies suggest that approximately 80% of the 25,000 human genes undergo alternative splicing. Alternative splicing may be associated with human diseases, particularly with cancer, but the molecular disease mechanisms are poorly understood. Convenient, novel methods for multiplexed detection of alternatively spliced transcripts are needed. METHODS: We devised a new approach for detecting splice variants based on a tag-microarray minisequencing system, originally developed for genotyping single-nucleotide polymorphisms. We established the system for multiplexed detection of 61 alternatively spliced transcripts in a panel of 19 cancer-related genes and used it to dissect the splicing patterns in cancer and endothelial cells. RESULTS: Our microarray system detected 82% of the splice variants screened for, including both simple and complex splice variants, in at least 1 of the leukemia cell types analyzed. The intraassay CV values for our method ranged from 0.01 to 0.34 (mean, 0.13) for 5 replicate measurements. Our system allowed semiquantitative comparison of the splicing patterns between the cell lines. Similar, but not identical, patterns of alternative splicing were observed among the leukemia cell lines. Size analysis of the PCR products subjected to the tag-array minisequencing system and real-time PCR with exon-junction probes verified the results from the microarray system. CONCLUSIONS: The microarray-based method is a robust and easily accessible tool for parallel detection of alternatively spliced transcripts of multiple genes. It can be used for studying alternative splicing in cancer progression and for following up drug treatment, and it may be a useful tool in clinical diagnostics for cancer and other disorders.

Alternative Splicing↗

PubMed2005

Alternative splicing and protein function.

BACKGROUND: Alternative splicing is a major mechanism of generating protein diversity in higher eukaryotes. Although at least half, and probably more, of mammalian genes are alternatively spliced, it was not clear, whether the frequency of alternative splicing is the same in different functional categories. The problem is obscured by uneven coverage of genes by ESTs and a large number of artifacts in the EST data. RESULTS: We have developed a method that generates possible mRNA isoforms for human genes contained in the EDAS database, taking into account the effects of nonsense-mediated decay and translation initiation rules, and a procedure for offsetting the effects of uneven EST coverage. Then we computed the number of mRNA isoforms for genes from different functional categories. Genes encoding ribosomal proteins and genes in the category "Small GTPase-mediated signal transduction" tend to have fewer isoforms than the average, whereas the genes in the category "DNA replication and chromosome cycle" have more isoforms than the average. Genes encoding proteins involved in protein-protein interactions tend to be alternatively spliced more often than genes encoding non-interacting proteins, although there is no significant difference in the number of isoforms of alternatively spliced genes. CONCLUSION: Filtering for functional isoforms satisfying biological constraints and accounting for uneven EST coverage allowed us to describe differences in alternative splicing of genes from different functional categories. The observations seem to be consistent with expectations based on current biological knowledge: less isoforms for ribosomal and signal transduction proteins, and more alternative splicing of interacting and cell cycle proteins.

Algorithms↗