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Results for “Alternative Splicing”

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PubMed2003

Identification of COL7A1 alternative splicing inserting 9 amino acid residues into the fibronectin type III linker domain.

Type VII collagen is the major component of anchoring fibrils within the cutaneous basement membrane zone. The large amino-terminal noncollagenous domain of type VII collagen interacts with various extracellular matrix proteins and contributes to the dermal-epidermal attachment. The purpose of this study was to detect alternative splicing of COL7A1 transcript encoding the noncollagenous 1 domain. The alternative splicing in this region may affect interactions of the noncollagenous 1 domain with extracellular matrix proteins and also dermal-epidermal adhesion. Thus we examined expression of the alternative splicing in situations relating to wound healing and skin remodeling that required dermal-epidermal binding and detachment. Amplification of overlapping cDNA from keratinocytes using reverse transcription-polymerase chain reaction identified alternative splicing, which was generated by a different exon 18 acceptor site 27 bp upstream from the common acceptor site. Expression of this alternatively spliced transcript differed among several cell types. The nine amino acid residues GPLTLPLSP from the 27 bp nucleotides were inserted into the linker of fibronectin type III domains. This insertion was suggested to contribute to flexibility of the linker of fibronectin type III domains and may affect the interactions between the noncollagenous 1 domain and extracellular matrix proteins. Treatment with transforming growth factor-beta 1, which is known to promote wound healing and skin remodeling, enhanced the expression of this 27 bp transcript. Furthermore, keratinocyte biopsies from the wound edge of patients with epithelizing skin ulcers showed a significant increase in the 27 bp transcript expression compared with normal keratinocytes from steady-state body sites. These results suggest that amino acid variation of this alternative splicing may have some role in dermal-epidermal adhesion, wound healing, and skin remodeling. To the best of our knowledge, this is the first evidence of alternative splice insertion of a small peptide into the linker region of the fibronectin type III domains, a common motif within modular proteins.

Alternative Splicing↗

PubMed1995

Alternative splicing of PSP94 (prostatic secretory protein of 94 amino acids) mRNA in prostate tissue.

While performing reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total mRNA from prostate cancer specimens, two forms of PSP94 cDNA were detected. RT-PCR products were analysed by Southern blotting and probing with exon-specific oligonucleotides. In the short form of PSP94 mRNA, designated as PSP57, exon III was found to be deleted. The two mRNA forms were confirmed by cloning and sequencing of the RT-PCR products and were found to result from alternative splicing. The alternatively spliced form, PSP57, was characterized by sequence analysis. PSP94 and PSP57 possess identical exons I and II, including identical secretion signal peptide and the 5' untranslated sequences. PSP57 has a frame-shifted exon IV and encodes a putative 57 amino acid protein with a novel, highly basic C-terminus of 41 amino acids. PSP57 mRNA was detected in other urogenital tissues (kidney, bladder) and in most tumor cell lines tested, but was not detectable in other tissues such as breast and lung. In prostate tumor cell lines, PSP57 mRNA was aberrantly spliced and localized in the nuclear fraction of the cell. Our results suggest the possible existence of a novel PSP protein that originates from alternative splicing of PSP94 mRNA in urogenital tissues.

Aged↗

PubMed1996

Alternative splicing of IgA Fc receptor (CD89) transcripts.

An alternatively spliced CD89 transcript is present in peripheral blood mononuclear cells (PBMC) and U937 cells. The alternatively spliced CD89 mRNA species lacks the exon 4 sequence, encompassing 288 nucleotides, that encodes the extracellular membrane-proximal immunoglobulin-like domain (EC2).

Alternative Splicing↗

PubMed2002

Function of quaking in myelination: regulation of alternative splicing.

Proteomic diversity is frequently achieved by alternative RNA-splicing events that can be fine-tuned in tissue-specific and developmentally regulated ways. Understanding this type of genetic regulation is compelling because of the extensive complexity of alternative splicing found in the nervous system. quaking (qk), one of the classical mouse dysmyelination mutants, is defective for the expression of myelin-associated glycoprotein (MAG), and the misregulation of MAG pre-mRNA alternative splicing is implicated as a causal factor. The qk locus encodes several RNA-binding proteins with heterogeneous nuclear ribonucleoprotein K-type homology, a characteristic of several known alternative splicing regulators. Here we test the nuclear-localized qk isoform (QKI-5) for its ability to regulate alternative splicing of MAG pre-mRNA in transient coexpression assays. QKI-5 exhibits properties of a negative regulator of MAG exon 12 alternative splicing. An intronic sequence element required for the repressive function and binding of QKI-5 is also identified. Direct evidence for irregularities in alternative splicing of MAG and other myelin protein transcripts in the qk mouse is demonstrated.

Alternative Splicing↗

PubMed2001

The neuron-specific RNA-binding protein ELAV regulates neuroglian alternative splicing in neurons and binds directly to its pre-mRNA.

Drosophila melanogaster neural-specific protein, ELAV, has been shown to regulate the neural-specific splicing of three genes: neuroglian (nrg), erect wing, and armadillo. Alternative splicing of the nrg transcript involves alternative inclusion of a 3'-terminal exon. Here, using a minigene reporter, we show that the nrg alternatively spliced intron (nASI) has all the determinants required to recreate proper neural-specific RNA processing seen with the endogenous nrg transcript, including regulation by ELAV. An in vitro UV cross-linking assay revealed that ELAV from nuclear extracts cross-links to four distinct sites along the 3200 nucleotide long nASI; one EXS is positioned at the polypyrimidine tract of the default 3' splice site. ELAV cross-linking sites (EXSs) have in common long tracts of (U)-rich sequence rather than a precise consensus; moreover, each tract has at least two 8/10U elements; their importance is validated by mutant transgene reporter analysis. Further, we propose criteria for ELAV target sequence recognition based on the four EXSs, sites within the nASI that are (U) rich but do not cross-link with ELAV, and predicted EXSs from a phylogenetic comparison with Drosophila virilis nASI. These results suggest that ELAV regulates nrg alternative splicing by direct interaction with the nASI.

Alternative Splicing↗

PubMed2000

TGF-beta superfamily members do not promote smooth muscle-specific alternative splicing, a late marker of vascular smooth muscle cell differentiation.

Smooth muscle (SM) specific alternate splicing of a number of genes is a late marker of the differentiated vascular smooth muscle cell (VSMC) phenotype and is one of the first differentiation characteristics to be lost during de-differentiation and in disease. An understanding of how this aspect of VSMC phenotype is regulated may provide insights into the earliest events of the atherosclerotic process. TGF-beta1 is a potent regulator of VSMC differentiation and can induce expression of SM-specific contractile proteins in both pluripotent stem cells and de-differentiated VSMCs. The purpose of this study was to test the hypothesis that members of the TGFbeta-superfamily can also effect SM-specific alternative splicing. Firstly, we established that SM-specific splicing of alpha-tropomyosin, vinculin and SM-myosin heavy chain (MHC) increases during rat fetal/neonatal development and is decreased in VSMCs following balloon-induced carotid injury in the rat. Treatment of cultured rat VSMCs with TGFbeta-superfamily members resulted in a significant reduction in the ratio of SM to non-muscle (NM) alpha-tropomyosin, but did not effect SM-specific alternative splicing of vinculin or SM-MHC. Treatment of pluripotent C3H10T1/2 cells with TGF-beta1, which increased SM differentiation marker expression, did not increase SM-specific alpha-tropomyosin splicing. Taken together, these results demonstrate differential regulation of SM-specific alternative splicing and indicate that although TGF-beta1 promotes VSMC differentiation marker expression, TGF-beta1 cannot act as the sole trigger of VSMC differentiation.

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PubMed2006

Phylogenetically widespread alternative splicing at unusual GYNGYN donors.

BACKGROUND: Splice donor sites have a highly conserved GT or GC dinucleotide and an extended intronic consensus sequence GTRAGT that reflects the sequence complementarity to the U1 snRNA. Here, we focus on unusual donor sites with the motif GYNGYN (Y stands for C or T; N stands for A, C, G, or T). RESULTS: While only one GY functions as a splice donor for the majority of these splice sites in human, we provide computational and experimental evidence that 110 (1.3%) allow alternative splicing at both GY donors. The resulting splice forms differ in only three nucleotides, which results mostly in the insertion/deletion of one amino acid. However, we also report the insertion of a stop codon in four cases. Investigating what distinguishes alternatively from not alternatively spliced GYNGYN donors, we found differences in the binding to U1 snRNA, a strong correlation between U1 snRNA binding strength and the preferred donor, over-represented sequence motifs in the adjacent introns, and a higher conservation of the exonic and intronic flanks between human and mouse. Extending our genome-wide analysis to seven other eukaryotic species, we found alternatively spliced GYNGYN donors in all species from mouse to Caenorhabditis elegans and even in Arabidopsis thaliana. Experimental verification of a conserved GTAGTT donor of the STAT3 gene in human and mouse reveals a remarkably similar ratio of alternatively spliced transcripts in both species. CONCLUSION: In contrast to alternative splicing in general, GYNGYN donors in addition to NAGNAG acceptors enable subtle protein variations.

Alternative Splicing↗

PubMed2005

Alternative splicing: a new drug target of the post-genome era.

Alternative splicing allows for the creation of multiple distinct mRNA transcripts from a given gene in a multicellular organism. Pre-mRNA splicing is catalyzed by a multi-molecular complex, including serine/arginine-rich (SR) proteins, which are highly phosphorylated in living cells, and thought to play crucial roles in spliceosomal formation and in the regulation of alternative splicing. Recently, reports of low molecular compounds, which alter splicing pattern of genes, have been accumulated. A benzothiazole compound TG003, a kinase inhibitor that targets Clk1 and Clk4, suppressed dissociation of nuclear speckles, altered the splicing patterns, and rescued the embryonic defects induced by excessive Clk activity. The emerging inhibitors of the signal transduction pathways regulating pre-mRNA alternative splicing may open the way to therapies against diseases caused by missplicing.

Alternative Splicing↗

PubMed1999

An alternatively spliced surfactant protein B mRNA in normal human lung: disease implication.

We identified an alternatively-spliced surfactant protein B (SP-B) mRNA from normal human lung with a 12 nt deletion at the beginning of exon 8. This deletion causes a loss of four amino acids in the SP-B precursor protein. Sequence comparison of the 3' splice sites reveals only one difference in the frequency of U/C in the 11 predominantly-pyrimidine nucleotide tract, 73% for the normal and 45% for the alternatively-spliced SP-B mRNA (77-99% for the consensus sequence). Analysis of SP-B mRNA in lung indicates that the abundance of the alternatively-spliced form is very low and varies among individuals. Although the relative abundance of the deletion form of SP-B mRNA remains constant among normal lungs, it is found with relatively higher abundance in the lungs of some individuals with diseases such as congenital alveolar proteinosis, respiratory distress syndrome, bronchopulmonary dysplasia, alveolar capillary dysplasia and hypophosphatasia. This observation points to the possibility that the alternative splicing is a potential regulatory mechanism of SP-B and may play a role in the pathogenesis of disease under certain circumstances.

Alternative Splicing↗

PubMed2001

Genome-wide detection of alternative splicing in expressed sequences of human genes.

We have identified 6201 alternative splice relationships in human genes, through a genome-wide analysis of expressed sequence tags (ESTs). Starting with approximately 2.1 million human mRNA and EST sequences, we mapped expressed sequences onto the draft human genome sequence and only accepted splices that obeyed the standard splice site consensus. A large fraction (47%) of these were observed multiple times, indicating that they comprise a substantial fraction of the mRNA species. The vast majority of the detected alternative forms appear to be novel, and produce highly specific, biologically meaningful control of function in both known and novel human genes, e.g. specific removal of the lysosomal targeting signal from HLA-DM beta chain, replacement of the C-terminal transmembrane domain and cytoplasmic tail in an FC receptor beta chain homolog with a different transmembrane domain and cytoplasmic tail, likely modulating its signal transduction activity. Our data indicate that a large proportion of human genes, probably 42% or more, are alternatively spliced, but that this appears to be observed mainly in certain types of molecules (e.g. cell surface receptors) and systemic functions, particularly the immune system and nervous system. These results provide a comprehensive dataset for understanding the role of alternative splicing in the human genome, accessible at http://www.bioinformatics.ucla.edu/HASDB.

Alternative Splicing↗

PubMed2003

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.

Alternative Splicing↗

PubMed2005

Detection and analysis of alternative splicing in the silkworm by aligning expressed sequence tags with the genomic sequence.

We identified 277 alternative splice forms in silkworm genes based on aligning expressed sequence tags with genomic sequences, using a transcript assembly program. A large fraction (74%) of these alternative splices are located in protein-coding regions and alter protein products, whereas only 26% are in untranslated regions. From the alternative splices located in protein-coding regions, some (43%) affect protein domains that bind various biological molecules. The vast majority of the detected alternative forms in this study appear to be novel, and potentially affect biologically meaningful control of function in silkworm genes. Our results indicate that alternative splicing in silkworm largely produces protein diversity and functional diversity, and is a widely used mechanism for regulating gene expression.

Alternative Splicing↗

PubMed2001

Distinctive features of Drosophila alternative splicing factor RS domain: implication for specific phosphorylation, shuttling, and splicing activation.

The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5' alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.

Alternative Splicing↗

PubMed2004

Stage-specific alternative splicing of the heat-shock transcription factor during the life-cycle of Schistosoma mansoni.

Stage-specific alternative splicing of the heat-shock transcription factor of Schistosoma mansoni (SmHSF) generates isoforms with structural diversity that may modulate the activity of SmHSF at different life-stages, and thus may regulate the expression of different genes at different developmental stages. RT-PCR, cloning and DNA-sequence analyses showed stage-specific alternative splicing inside the DNA-binding domain (DBD) involving introns I1 and I2, and beyond the DBD involving introns I4a and I7. Retention of introns I2 and I4 would inactivate SmHSF since they contain termination codons. Retention of intron I1 would add 11 amino acids inside the DBD and may change the DNA-binding specificity of SmHSF; intron I7 would add 13 amino acids to the effector region of HSF. Retention of introns was more pronounced in cercariae (larval stage living in water) than in adult worms (parasitic form in mammals). The isoforms were expressed in bacteria, but functional evaluation was not feasible, because only the isoform lacking introns was soluble while isoforms with introns were insoluble. However, stage-specific alternative splicing that changed HSF function in vivo was evidenced in intact cercariae. The cercarial SmHSF mRNA was enriched with introns I2 and I4a that contain termination codons. Therefore, translation of the SmHSF mRNA was impaired, and the SmHSF protein was undetectable. Consequently, the HSP70 gene could not be transcribed, and the HSP70 mRNA was missing. Alternative splicing was observed for short DNA segments (33-45 bp) bound by splice signals, located in the coding region. These are not bona fida exons since they are not flanked by introns. Yet, they are not regular introns since they are often found in mature mRNA. Alternative splicing of these DNA segments caused structural diversity that could modulate the function of the gene product.

Alternative Splicing↗

PubMed1998

Identification of intron and exon sequences involved in alternative splicing of insulin receptor pre-mRNA.

The insulin receptor exists as two isoforms, A and B, that result from alternative splicing of exon 11 in the primary transcript. We have shown previously that the alternative splicing is developmentally and hormonally regulated. Consequently, these studies were instigated to identify sequences within the primary RNA transcript that regulate the alternative splicing. Minigenes containing exons 10, 11, and 12 and the intervening introns were constructed and transfected into HepG2 cells, which contain both isoforms of the insulin receptor. The cells were able to splice the minigene transcript to give both A (- exon 11) and B-like (+ exon 11) RNAs. A series of internal deletions within intron 10 were tested for their ability to give A and B RNAs. Intron 10 contained two sequences that modulated exon 11 inclusion; a 48-nucleotide purine-rich sequence at the 5' end of intron 10 that functions as a splicing enhancer and causes an increase in exon 11 inclusion, and a 43-nucleotide sequence at the 3' end of intron 10 upstream of the branch point sequence that favors skipping of exon 11. Increasing the length of the polypyrimidine tract at the 3' end of intron 10 caused exon 11 to be spliced constitutively, indicating that a weak splice site is required for alternative splicing. Finally, point mutations, insertions, and deletions within exon 11 itself were able to regulate inclusion of the exon both positively and negatively.

Alternative Splicing↗

PubMed2002

Alu-containing exons are alternatively spliced.

Alu repetitive elements are found in approximately 1.4 million copies in the human genome, comprising more than one-tenth of it. Numerous studies describe exonizations of Alu elements, that is, splicing-mediated insertions of parts of Alu sequences into mature mRNAs. To study the connection between the exonization of Alu elements and alternative splicing, we used a database of ESTs and cDNAs aligned to the human genome. We compiled two exon sets, one of 1176 alternatively spliced internal exons, and another of 4151 constitutively spliced internal exons. Sixty one alternatively spliced internal exons (5.2%) had a significant BLAST hit to an Alu sequence, but none of the constitutively spliced internal exons had such a hit. The vast majority (84%) of the Alu-containing exons that appeared within the coding region of mRNAs caused a frame-shift or a premature termination codon. Alu-containing exons were included in transcripts at lower frequencies than alternatively spliced exons that do not contain an Alu sequence. These results indicate that internal exons that contain an Alu sequence are predominantly, if not exclusively, alternatively spliced. Presumably, evolutionary events that cause a constitutive insertion of an Alu sequence into an mRNA are deleterious and selected against.

Alternative Splicing↗

PubMed2003

Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss.

One of the most interesting opportunities in comparative genomics is to compare not only genome sequences but additional phenomena, such as alternative splicing, using orthologous genes in different genomes to find similarities and differences between organisms. Recently, genomics studies have suggested that 40-60% of human genes are alternatively spliced and have catalogued up to 30,000 alternative splice relationships in human genes. Here we report an analysis of 9,434 orthologous genes in human and mouse, which indicates that alternative splicing is associated with a large increase in frequency of recent exon creation and/or loss. Whereas most exons in the mouse and human genomes are strongly conserved in both genomes, exons that are only included in alternative splice forms (as opposed to the constitutive or major transcript form) are mostly not conserved and thus are the product of recent exon creation or loss events. A similar comparison of orthologous exons in rat and human validates this pattern. Although this says nothing about the complex question of adaptive benefit, it does indicate that alternative splicing in these genomes has been associated with increased evolutionary change.

Alternative Splicing↗

PubMed2002

Differential subcellular localization of the two alternatively spliced isoforms of the Kv3.1 potassium channel subunit in brain.

Voltage-gated K(+) channels containing pore-forming subunits of the Kv3 subfamily have specific roles in the fast repolarization of action potentials and enable neurons to fire repetitively at high frequencies. Each of the four known Kv3 genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K(+) channel subunits differing only in their C-terminal sequence. The alternative splicing does not affect the electrophysiological properties of the channels, and its physiological role is unknown. It has been proposed that one of the functions of the alternative splicing of Kv3 genes is to produce subunit isoforms with differential subcellular membrane localizations in neurons and differential modulation by signaling pathways. We investigated the role of the alternative splicing of Kv3 subunits in subcellular localization by examining the brain distribution of the two alternatively spliced versions of the Kv3.1 gene (Kv3.1a and Kv3.1b) with antibodies specific for the alternative spliced C-termini. Kv3.1b proteins were prominently expressed in the somatic and proximal dendritic membrane of specific neuronal populations in the mouse brain. The axons of most of these neurons also expressed Kv3.1b protein. In contrast, Kv3.1a proteins were prominently expressed in the axons of some of the same neuronal populations, but there was little to no Kv3.1a protein expression in somatodendritic membrane. Exceptions to this pattern were seen in two neuronal populations with unusual targeting of axonal proteins, mitral cells of the olfactory bulb, and mesencephalic trigeminal neurons, which expressed Kv3.1a protein in dendritic and somatic membrane, respectively. The results support the hypothesis that the alternative spliced C-termini of Kv3 subunits regulate their subcellular targeting in neurons.

Alternative Splicing↗