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Results for “Alternative Splicing”

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PubMed2002

A splicing silencer that regulates smooth muscle specific alternative splicing is active in multiple cell types.

Alternative splicing of alpha-tropomyosin (alpha-TM) involves mutually exclusive selection of exons 2 and 3. Selection of exon 2 in smooth muscle (SM) cells is due to inhibition of exon 3, which requires both binding sites for polypyrimidine tract-binding protein as well as UGC (or CUG) repeat elements on both sides of exon 3. Point mutations or substitutions of the UGC-containing upstream regulatory element (URE) with other UGC elements disrupted the alpha-TM splicing pattern in transfected cells. Multimerisation of the URE caused enhanced exon skipping in SM and various non-SM cells. In the presence of multiple UREs the degree of splicing regulation was decreased due to the high levels of exon skipping in non-SM cell lines. These results suggest that the URE is not an intrinsically SM- specific element, but that its functional strength is fine tuned to exploit differences in the activities of regulatory factors between SM and other cell types. Co-transfection of tropomyosin reporters with members of the CUG-binding protein family, which are candidate URE-binding proteins, indicated that these factors do not mediate repression of tropomyosin exon 3.

Alternative Splicing↗

PubMed2001

SC35 plays a role in T cell development and alternative splicing of CD45.

Molecular diversity via alternative splicing is important for cellular function and development. SR proteins are strong candidate regulators of alternative splicing because they can modulate splice site selection. However, endogenous substrates for SR proteins are largely unknown, and their roles as splicing regulators in vertebrate development are unclear. Here we report that Cre-mediated conditional deletion of the prototypical SR protein SC35 in the thymus causes a defect in T cell maturation. Deletion of SC35 alters alternative splicing of CD45, a receptor tyrosine phosphatase known to be regulated by differential splicing during thymocyte development and activation. This study establishes a model to address the function of SR proteins in physiological settings and reveals a critical role of SC35 in a T cell-specific regulated splicing pathway.

Alternative Splicing↗

PubMed2005

A cross-talk between RNA splicing and signaling pathway alters Fas gene expression at post-transcriptional level: alternative splicing of Fas mRNA in the leukemic U937 cells.

It is now widely accepted that alternative splicing is a mechanism that is responsible for generating protein complexity at low genetic cost. However, little is known about molecular mechanisms that govern alternative splicing of key apoptotic regulators. Here we investigate the effect of pro-apoptotic stimuli on alternative splicing of Fas mRNA by means of reverse transcription-polymerase chain reaction (RT-PCR). Exposure of U937 cells to etoposide, staurosporine, pacritaxel, or cyclohexamide promoted the appearance of the splice variant, which retained the 152-base-pair intron 5. Pretreatment with calyculin A, an inhibitor of protein phosphatase-1 (PP-1) as well as fumonisin B1, an inhibitor of ceramide synthase, prevented etoposide-induced alternative splicing of Fas mRNA. Our data demonstrate that cross-talk between RNA splicing and signaling pathways through endogenous ceramide synthesis and subsequent phosphatase activation is a mechanism that modifies Fas gene expression at the posttranscriptional level.

Alternative Splicing↗

PubMed2003

Cis-acting intronic elements that regulate cartilage-specific alternative splicing of the type II collagen (Col2) pre-mRNA lie at or near splice site junction sequences flanking exon 2 of the gene.

UNLABELLED: Knowledge of the cis-acting elements is required for identifying trans-acting splicing factors underlying cartilage-specific alternative splicing of Col2 pre-mRNA. By performing desired deletions in the mouse Col2 pre-mRNA, location of the intronic cis-acting elements was narrowed down to be at or near splice-junction sequences flanking exon 2 of the gene. INTRODUCTION: Type II collagen (Col2) pre-mRNA undergoes cartilage-specific alternative splicing involving exon 2 during chondrocyte differentiation. Thus, the trans-acting protein factors that regulate the splicing are associated with the differentiation of chondrocytes. Knowledge of the cognate cis-acting elements is necessary to eventually identify the trans-acting factors. MATERIALS AND METHODS: To localize the cis-acting sequences, we created several deletions within a minigene containing exon 1 to exon 4 of mouse Col 2 gene and evaluated alternative splicing of the resulting pre-mRNAs in ATDC5 cells, a model of insulin-stimulated chondrocyte differentiation. The first deletion reduced intron 1 from 3799 to 259 bp, the second reduced intron 2 from 1108 to 94 bp, the third combined the above two deletions, and the fourth was derived from the third by removing intron 3 and exon 4. ATDC5 cells harboring these constructs were cultured for up to 21 days with or without insulin. Alternative splicing was evaluated by determining the ratio of Col2B (lacks exon 2) to Col2A (has exon 2) RNAs by reverse transcription-polymerase chain reaction. RESULTS: The deletion in intron 1 had no effect on the alternative splicing while other deletions affected splicing (demonstrated by the presence of splicing intermediates) in cells cultured without insulin or with insulin for 1 week. The splicing intermediates were not seen from any construct when cells were cultured longer (14-21 days) with insulin. CONCLUSION: These results show that the 259-bp intron 1, the 94-bp intron 2, and exon 2 sequences retained in the fourth construct provide cis-acting signal sufficient for insulin-induced cartilage-specific alternative splicing of Col2 pre-mRNA.

Alternative Splicing↗

PubMed2000

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

Alternative Splicing↗

PubMed2004

Genome-wide detection of alternative splicing in expressed sequences using partial order multiple sequence alignment graphs.

We present a method for high-throughput alternative splicing detection in expressed sequence data. This method effectively copes with many of the problems inherent in making inferences about splicing and alternative splicing on the basis of EST sequences, which in addition to being fragmentary and full of sequencing errors, may also be chimeric, misoriented, or contaminated with genomic sequence. Our method, which relies both on the Partial Order Alignment (POA) program for constructing multiple sequence alignments, and its Heaviest Bundling function for generating consensus sequences, accounts for the real complexity of expressed sequence data by building and analyzing a single multiple sequence alignment containing all of the expressed sequences in a particular cluster aligned to genomic sequence. We illustrate application of this method to human UniGene Cluster Hs.1162, which contains expressed sequences from the human HLA-DMB gene. We have used this method to generate databases, published elsewhere, of splices and alternative splicing relationships for the human, mouse and rat genomes. We present statistics from these calculations, as well as the CPU time for running our method on expressed sequence clusters of varying size, to verify that it truly scales to complete genomes.

Alternative Splicing↗

PubMed2005

RASE: recognition of alternatively spliced exons in C.elegans.

MOTIVATION: Eukaryotic pre-mRNAs are spliced to form mature mRNA. Pre-mRNA alternative splicing greatly increases the complexity of gene expression. Estimates show that more than half of the human genes and at least one-third of the genes of less complex organisms, such as nematodes or flies, are alternatively spliced. In this work, we consider one major form of alternative splicing, namely the exclusion of exons from the transcript. It has been shown that alternatively spliced exons have certain properties that distinguish them from constitutively spliced exons. Although most recent computational studies on alternative splicing apply only to exons which are conserved among two species, our method only uses information that is available to the splicing machinery, i.e. the DNA sequence itself. We employ advanced machine learning techniques in order to answer the following two questions: (1) Is a certain exon alternatively spliced? (2) How can we identify yet unidentified exons within known introns? RESULTS: We designed a support vector machine (SVM) kernel well suited for the task of classifying sequences with motifs having positional preferences. In order to solve the task (1), we combine the kernel with additional local sequence information, such as lengths of the exon and the flanking introns. The resulting SVM-based classifier achieves a true positive rate of 48.5% at a false positive rate of 1%. By scanning over single EST confirmed exons we identified 215 potential alternatively spliced exons. For 10 randomly selected such exons we successfully performed biological verification experiments and confirmed three novel alternatively spliced exons. To answer question (2), we additionally used SVM-based predictions to recognize acceptor and donor splice sites. Combined with the above mentioned features we were able to identify 85.2% of skipped exons within known introns at a false positive rate of 1%. AVAILABILITY: Datasets, model selection results, our predictions and additional experimental results are available at http://www.fml.tuebingen.mpg.de/~raetsch/RASE SUPPLEMENTARY INFORMATION: http://www.fml.tuebingen.mpg.de/raetsch/RASE.

Algorithms↗

PubMed1996

Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.

Alternative Splicing↗

PubMed2003

Alternative splicing of the ovine CFTR gene.

Alternative splicing of the human CFTR gene was studied previously and shown not to generate functional CFTR-like chloride ion channels. However, it is possible that some of the alternatively spliced forms may encode CFTR proteins with different functions. The ovine CFTR gene is very similar to the human gene and has regulatory mechanisms in common. To evaluate whether the alternatively spliced forms of human CFTR are conserved in the sheep, the splice forms of the ovine CFTR gene were examined. A transcript lacking exon 9 was observed in the sheep, but unlike the human exon 9-transcript, it did not result from a polymorphic intron 8 splice acceptor site. Sheep CFTR transcripts lacking exon 17b were seen and have also been described in the human. Transcripts lacking 98 bp of the 5' end of exon 13, the whole of exon 13, and both exons 14b and 15 respectively were seen in sheep but have not been reported in human. Splice site donor and acceptor sequences were isolated, and alternative transcripts were shown to result from a combination of aberrant sites and competition of 5' splice donor sequences.

Alternative Splicing↗

PubMed2004

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.

Alternative Splicing↗

PubMed2003

Increase of functional diversity by alternative splicing.

A large-scale analysis of protein isoforms arising from alternative splicing shows that alternative splicing tends to insert or delete complete protein domains more frequently than expected by chance, whereas disruption of domains and other structural modules is less frequent. If domain regions are disrupted, the functional effect, as predicted from 3D structure, is frequently equivalent to removal of the entire domain. Also, short alternative splicing events within domains, which might preserve folded structure, target functional residues more frequently than expected. Thus, it seems that positive selection has had a major role in the evolution of alternative splicing.

Alternative Splicing↗

PubMed2005

Integrating alternative splicing detection into gene prediction.

BACKGROUND: Alternative splicing (AS) is now considered as a major actor in transcriptome/proteome diversity and it cannot be neglected in the annotation process of a new genome. Despite considerable progresses in term of accuracy in computational gene prediction, the ability to reliably predict AS variants when there is local experimental evidence of it remains an open challenge for gene finders. RESULTS: We have used a new integrative approach that allows to incorporate AS detection into ab initio gene prediction. This method relies on the analysis of genomically aligned transcript sequences (ESTs and/or cDNAs), and has been implemented in the dynamic programming algorithm of the graph-based gene finder EuGENE. Given a genomic sequence and a set of aligned transcripts, this new version identifies the set of transcripts carrying evidence of alternative splicing events, and provides, in addition to the classical optimal gene prediction, alternative optimal predictions (among those which are consistent with the AS events detected). This allows for multiple annotations of a single gene in a way such that each predicted variant is supported by a transcript evidence (but not necessarily with a full-length coverage). CONCLUSIONS: This automatic combination of experimental data analysis and ab initio gene finding offers an ideal integration of alternatively spliced gene prediction inside a single annotation pipeline.

Algorithms↗

PubMed2002

Regulation of corticotropin-releasing hormone type 2 receptors by multiple promoters and alternative splicing: identification of multiple splice variants.

We demonstrate that multiple promoters and alternate splicing regulate expression of the human CRH receptor type 2 (CRHR2) gene. We show that flanking regions to the first exons drive promoter activity in both endogenously and nonendogenously expressing cell lines. Putative promoter elements have been identified that are conserved between species, including the comparison of CRHR2gamma in nonhuman primates that was previously known only in humans, which may be responsible for subtype tissue specific regulation. We have identified novel transcripts produced by alternate splicing of the first exon of CRHR2beta (beta1a) with various combinations of the 5' exons including a novel exon (beta1c) spliced to the common exons. The 5' structure of the gene permits many other combinations of alternate splicing that may arise as part of a regulatory mechanism controlling functional receptor expression. The 5'-untranslated region of the first exons has been extended; and 3' acceptor sites identified within the 5' untranslated region of CRHR2gamma and CRHR2alpha are used during alternate splicing of CRHR2beta upstream exons. This has important implications because various reports on the expression of CRHR2gamma and CRHR2alpha have been unable to discriminate between the functional receptor and CRHR2beta alternate splice variants. Only the described sequences upstream of the 3' splice site are unique to CRHR2gamma and CRHR2alpha.

5' Untranslated Regions↗

PubMed1994

Evolutionary conservation of the structure and expression of alternatively spliced Ultrabithorax isoforms from Drosophila.

In Drosophila melanogaster, alternatively spliced mRNAs from the homeotic gene Ultrabithorax (Ubx) encode a family of structurally distinct homeoprotein isoforms. The developmentally regulated expression patterns of these isoforms suggest that they have specialized stage- and tissue-specific functions. To evaluate the functional importance of UBX isoform diversity and gain clues to the mechanism that regulates processing of Ubx RNAs, we have investigated whether the Ubx RNAs of other insects undergo similar alternative splicing. We have isolated and characterized Ubx cDNA fragments from D. melanogaster, Drosophila pseudoobscura, Drosophila hydei and Drosophila virilis, species separated by as much as 60 million years of evolution, and have found that three aspects of Ubx RNA processing have been conserved. (1) These four species exhibit identical patterns of optional exon use in a region adjacent to the homeodomain. (2) These four species produce the same family of UBX protein isoforms with identical amino acid sequences in the optional exons, even though the common amino-proximal region has undergone substantial divergence. The nucleotide sequences of the optional exons, including third positions of rare codons, have also been conserved strongly, suggesting functional constraints that are not limited to coding potential. (3) The tissue- and stage-specific patterns of expression of different UBX isoforms are identical among these Drosophila species, indicating that the developmental regulation of the alternative splicing events has also been conserved. These findings argue for an important role of alternative splicing in Ubx function. We discuss the implications of these results for models of UBX protein function and the mechanism of alternative splicing.

Alternative Splicing↗

PubMed2004

Accurate identification of alternatively spliced exons using support vector machine.

MOTIVATION: Alternative splicing is a major component of the regulatory action on mammalian transcriptomes. It is estimated that over half of all human genes have more than one splice variant. Previous studies have shown that alternatively spliced exons possess several features that distinguish them from constitutively spliced ones. Recently, we have demonstrated that such features can be used to distinguish alternative from constitutive exons. In the current study, we used advanced machine learning methods to generate robust classifier of alternative exons. RESULTS: We extracted several hundred local sequence features of constitutive as well as alternative exons. Using feature selection methods we find seven attributes that are dominant for the task of classification. Several less informative features help to slightly increase the performance of the classifier. The classifier achieves a true positive rate of 50% for a false positive rate of 0.5%. This result enables one to reliably identify alternatively spliced exons in exon databases that are believed to be dominated by constitutive exons.

Algorithms↗

PubMed2004

Mammary epithelial-mesenchymal interaction regulates fibronectin alternative splicing via phosphatidylinositol 3-kinase.

The way alternative splicing is regulated within tissues is not understood. A relevant model of this process is provided by fibronectin, an important extracellular matrix protein that plays a key role in cell adhesion and migration and contains three alternatively spliced regions known as EDI, EDII, and IIICS. We used a cell culture system to simulate mammary epithelial-stromal communication, a process that is crucial for patterning and function of the mammary gland, and studied the effects of extracellular signals on the regulation of fibronectin pre-mRNA alternative splicing. We found that soluble factors from a mammary mesenchymal cell-conditioned medium, as well as the growth factors HGF/SF (hepatocyte growth factor/scatter factor), KGF (keratinocyte growth factor), and aFGF (acidic fibroblast growth factor), stimulate EDI and IIICS but not EDII inclusion into fibronectin mRNA in the mammary epithelial cell line SCp2, favoring fibronectin isoforms associated with proliferation, migration, and tissue remodeling. We explored the signaling pathways involved in this regulation and found that the mammary mesenchymal cell-conditioned medium and HGF/SF act through a phosphatidylinositol 3-kinase-dependent cascade to alter fibronectin alternative splicing. This splicing regulation is independent from promoter structure and de novo protein synthesis but does require two exonic elements within EDI. These results shed light on how extracellular stimuli are converted into changes in splicing patterns.

Alternative Splicing↗

PubMed2002

Half pint regulates alternative splice site selection in Drosophila.

Alternative splicing is used by metazoans to increase protein diversity and to alter gene expression during development. However, few factors that control splice site choice in vivo have been identified. Here we describe a factor, Half pint (Hfp), that regulates RNA splicing in Drosophila. Females harboring hypomorphic mutations in hfp lay short eggs and show defects in germline mitosis, nuclear morphology, and RNA localization during oogenesis. We find that hfp encodes the Drosophila ortholog of human PUF60 and functions in both constitutive and alternative splicing in vivo. In particular, hfp mutants display striking defects in the developmentally regulated splicing of ovarian tumor (otu). Furthermore, transgenic expression of the missing otu splice form can rescue the ovarian phenotypes of hfp.

Alternative Splicing↗

PubMed1992

Expression of alternatively spliced human T-lymphotropic virus type I pX mRNA in infected cell lines and in primary uncultured cells from patients with adult T-cell leukemia/lymphoma and healthy carriers.

Although human T-cell lymphotropic virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia/lymphoma (ATL), the role of viral gene expression in the progression to and maintenance of the leukemic state in vivo is unclear because of the inability of most previous studies to readily detect HTLV-I RNA in infected individuals. By using the reverse transcriptase-polymerase chain reaction, we detected spliced messages for the HTLV-I pX regulatory genes in primary uncultured cells from ATL patients and healthy asymptomatic carriers. In addition to the expected doubly spliced pX message, three alternatively spliced mRNAs were demonstrated (pX delta 17, pX-p21rex, and pX-orfII mRNAs, where orf = open reading frame). The same splice sites were shown in the messages from uncultured ATL cells and from the HTLV-I-producing C10/MJ cell line. Alternatively spliced pX mRNAs have the potential to code for known and putative pX gene products. Among the transcripts is a monocistronic mRNA likely to code for p21rex (pX-p21rex mRNA). Since alternative splicing of HTLV-I pX mRNA can be found in primary uncultured cells, it is likely to have a functional significance in vivo. This suggests possible roles for HTLV-I gene expression in the progression to and maintenance of ATL, as well as in the phase preceding it.

Base Sequence↗