Biomedical subjects

Stefan Stamm

Publications and source records attributed to Stefan Stamm.

At least 19 recordsLinked to original sources

PubMed2006

WT1 interacts with the splicing protein RBM4 and regulates its ability to modulate alternative splicing in vivo.

Wilm's tumor protein 1 (WT1), a protein implicated in various cancers and developmental disorders, consists of two major isoforms: WT1(-KTS), a transcription factor, and WT1(+KTS), a post-transcriptional regulator that binds to RNA and can interact with splicing components. Here we show that WT1 interacts with the novel splicing regulator RBM4. Each protein was found to colocalize in nuclear speckles and to cosediment with supraspliceosomes in glycerol gradients. RBM4 conferred dose-dependent and cell-specific regulation of alternative splicing of pre-mRNAs transcribed from several reporter genes. We found that overexpressed WT1(+KTS) abrogated this effect of RBM4 on splice-site selection, whereas WT1(-KTS) did not. We conclude that the (+KTS) form of WT1 is able to inhibit the effect of RBM4 on alternative splicing.

Alternative Splicing↗

PubMed2006

Splicing factor Tra2-beta1 is specifically induced in breast cancer and regulates alternative splicing of the CD44 gene.

The human CD44 gene undergoes extensive alternative splicing of multiple variable exons positioned in a cassette in the middle of the gene. Expression of alternative exons is often restricted to certain tissues and could be associated with tumor progression and metastasis of several human malignancies, including breast cancer. Exon v4 contains multiple copies of a C/A-rich exon enhancer sequence required for optimal inclusion of the exon and binding to the nucleic acid-binding proteins YB-1 and human Tra2-beta1. Here, we show that hTra2-beta1, a member of the extended family of serine/arginine-rich (SR) splicing factors, enhances the in vivo inclusion of CD44 exons v4 and v5. It increased inclusion of exons v4 and v5 and acted synergistically with YB-1. Activation required the C/A-rich enhancer within exon v4. Several other SR proteins had none or only a slight effect on CD44 exon inclusion. In contrast, SC35 inhibited exon usage and antagonized the effects of Tra2 or YB-1. In a matched pair analysis of human breast cancers and their corresponding nonpathologic tissue controls, we found a significant induction of Tra2-beta1 in invasive breast cancer, both on the RNA and protein levels. Together with our functional data, these results suggest an important role for Tra2-beta1 in breast cancer. Induction of this splicing factor might be responsible for splicing of CD44 isoforms associated with tumor progression and metastasis.

Alternative Splicing↗

PubMed2006

ASD: a bioinformatics resource on alternative splicing.

Alternative splicing is an important regulatory mechanism of mammalian gene expression. The alternative splicing database (ASD) consortium is systematically collecting and annotating data on alternative splicing. We present the continuation and upgrade of the ASD [T. A. Thanaraj, S. Stamm, F. Clark, J. J. Riethoven, V. Le Texier, J. Muilu (2004) Nucleic Acids Res. 32, D64-D69] that consists of computationally and manually generated data. Its largest parts are AltSplice, a value-added database of computationally delineated alternative splicing events. Its data include alternatively spliced introns/exons, events, isoform splicing patterns and isoform peptide sequences. AltSplice data are generated by examining gene-transcript alignments. The data are annotated for various biological features including splicing signals, expression states, (SNP)-mediated splicing and cross-species conservation. AEdb forms the manually curated component of ASD. It is a literature-based data set containing sequence and properties of alternatively spliced exons, functional enumeration of observed splicing events, characterization of observed splicing regulatory elements, and a collection of experimentally clarified minigene constructs. ASD includes a workbench, which is an analysis tool that enables users to carry out splicing related analysis such as characterization of introns for various splicing signals, identification of splicing regulatory elements on a given RNA sequence, prediction of putative exons and prediction of putative translation start codons. The different ASD modules are integrated and can be accessed through user-friendly interfaces and visualization tools. ASD data has been integrated with Ensembl genome annotation project as a Distributed Annotation System (DAS) resource and can be viewed on Ensembl genome browser. The ASD resource is presented at (http://www.ebi.ac.uk/asd).

Alternative Splicing↗

PubMed2006

Pre-mRNA missplicing as a cause of human disease.

Regulated alternative splice site selection emerges as one of the most important mechanisms to control the expression of genetic information in humans. It is therefore not surprising that a growing number of diseases are either associated with or caused by changes in alternative splicing. These diseases can be caused by mutation in regulatory sequences of the pre-mRNA or by changes in the concentration of trans-acting factors. The pathological expression of mRNA isoforms can be treated by transferring nucleic acids derivatives into cells that interfere with sequence elements on the pre-mRNA, which results in the desired splice site selection. Recently, a growing number of low molecular weight drugs have been discovered that influence splice site selection in vivo. These findings prove the principle that diseases caused by missplicing events could eventually be cured.

Base Sequence↗

PubMed2005

The alternative splicing of tau exon 10 and its regulatory proteins CLK2 and TRA2-BETA1 changes in sporadic Alzheimer's disease.

Pathological inclusions containing fibrillar aggregates of hyperphosphorylated tau protein are a characteristic feature in tauopathies, which include Alzheimer's disease (AD). Tau is a microtubule-associated protein whose transcript undergoes alternative splicing in the brain. Exon 10 encodes one of four microtubule-binding repeats. Exon 10 inclusion gives rise to tau protein isoforms containing four microtubule-binding repeats (4R) whereas exclusion leads to isoforms containing only three repeats (3R). The ratio between 3R and 4R isoforms is tightly controlled via alternative splicing in the human adult nervous system and distortion of this balance results in neurodegeneration. Previous studies showed that several splicing regulators, among them hTRA2-beta1 and CLK2, regulate exon 10 alternative splicing. Like most splicing factors, htra2-beta and clk2 pre-mRNAs are regulated by alternative splicing. Here, we investigated whether human postmortem brain tissue of AD patients reveal differences in alternative splicing patterns of the tau, htra2-beta, presenilin 2 and clk2 genes when compared with age-matched controls. We found that the splicing patterns of all four genes are altered in affected brain areas of sporadic AD patients. In these affected areas, the amount of mRNAs of tau isoforms including exon 10, the htra2-beta1 isoform and an inactive form of clk2 are significantly increased. These findings suggest that a misregulation of alternative splicing seems to contribute to sporadic AD.

Aged↗

PubMed2005

The snoRNA HBII-52 regulates alternative splicing of the serotonin receptor 2C.

The Prader-Willi syndrome is a congenital disease that is caused by the loss of paternal gene expression from a maternally imprinted region on chromosome 15. This region contains a small nucleolar RNA (snoRNA), HBII-52, that exhibits sequence complementarity to the alternatively spliced exon Vb of the serotonin receptor 5-HT(2C)R. We found that HBII-52 regulates alternative splicing of 5-HT(2C)R by binding to a silencing element in exon Vb. Prader-Willi syndrome patients do not express HBII-52. They have different 5-HT(2C)R messenger RNA (mRNA) isoforms than healthy individuals. Our results show that a snoRNA regulates the processing of an mRNA expressed from a gene located on a different chromosome, and the results indicate that a defect in pre-mRNA processing contributes to the Prader-Willi syndrome.

Alternative Splicing↗

PubMed2005

Identification, expression analysis, genomic organization and cellular location of a novel protein with a RhoGEF domain.

In this study we describe the identification and characterization of a novel cytosolic protein of the guanine exchange factor (GEF) family. The human cDNA corresponds to predicted human protein FLJ00128/FLJ10357 located on chromosome 14q11.2. The deduced protein sequence contains in its C-terminus a RhoGEF domain followed by a pleckstrin domain. Its N-terminus, central region and RhoGEF/pleckstrin domain are homologous to the recently identified zebrafish Quattro protein, which is involved in morphogenetic movements mediated by the actin cytoskeleton. Based on the homology of our protein's RhoGEF domain to the RhoGEF domains of Trio, Duo and Duet and its homology with Quattro, we named it Solo. The Solo mRNA is ubiquitously expressed but enriched in brain, its expression peaks perinatally and it undergoes extensive alternative splicing. In both myoblasts and neuroblastoma cells, the Solo protein is concentrated around the nucleus.

Amino Acid Sequence↗

PubMed2005

Tau exons 2 and 10, which are misregulated in neurodegenerative diseases, are partly regulated by silencers which bind a SRp30c.SRp55 complex that either recruits or antagonizes htra2beta1.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 2 modulates the tau N-terminal domain, which interacts with the axonal membrane. Exon 10 codes for a microtubule binding domain, increasing the affinity of tau for microtubules. Both exons are excluded from fetal brain, but their default behavior is inclusion, suggesting that silencers are involved in their regulation. Exon 2 is significantly reduced in myotonic dystrophy type 1, whose symptoms include dementia. Mutations that affect exon 10 splicing cause frontotemporal dementia (FTDP). In this study, we investigated three regulators of exon 2 and 10 splicing: serine/arginine-rich (SR) proteins SRp55, SRp30c, and htra2beta1. The first two inhibit both exons; htra2beta1 inhibits exon 2 but activates exon 10. By deletion analysis, we identified splicing silencers located at the 5' end of each exon. Furthermore, we demonstrated that SRp30c and SRp55 bind to both silencers and to each other. In exon 2, htra2beta1 binds to the inhibitory heterodimer through its RS1 domain but not to exon 2, whereas in exon 10 the heterodimer may sterically interfere with htra2beta1 binding to a purine-rich enhancer (defined by FTDP mutation E10-Delta5 = Delta280K) directly downstream of the silencer. Increased exon 10 inclusion in FTDP mutant ENH (N279K) may arise from abolishing SRp30c binding. Also, htra2beta3, a naturally occurring variant of htra2beta1, no longer inhibits exon 2 splicing but can partially rescue splicing of exon 10 in FTDP mutation E10-Delta5. This work provides interesting insights into the splicing regulation of the tau gene.

Animals↗

PubMed2004

Function of alternative splicing.

Alternative splicing is one of the most important mechanisms to generate a large number of mRNA and protein isoforms from the surprisingly low number of human genes. Unlike promoter activity, which primarily regulates the amount of transcripts, alternative splicing changes the structure of transcripts and their encoded proteins. Together with nonsense-mediated decay (NMD), at least 25% of all alternative exons are predicted to regulate transcript abundance. Molecular analyses during the last decade demonstrate that alternative splicing determines the binding properties, intracellular localization, enzymatic activity, protein stability and posttranslational modifications of a large number of proteins. The magnitude of the effects range from a complete loss of function or acquisition of a new function to very subtle modulations, which are observed in the majority of cases reported. Alternative splicing factors regulate multiple pre-mRNAs and recent identification of physiological targets shows that a specific splicing factor regulates pre-mRNAs with coherent biological functions. Therefore, evidence is now accumulating that alternative splicing coordinates physiologically meaningful changes in protein isoform expression and is a key mechanism to generate the complex proteome of multicellular organisms.

Alternative Splicing↗

PubMed2004

The intranuclear localization and function of YT521-B is regulated by tyrosine phosphorylation.

YT521-B is a ubiquitously expressed nuclear protein that changes alternative splice site usage in a concentration dependent manner. YT521-B is located in a dynamic nuclear compartment, the YT body. We show that YT521-B is tyrosine phosphorylated by c-Abl in the nucleus. The protein shuttles between nucleus and cytosol, where it can be phosphorylated by c-Src or p59(fyn). Tyrosine phosphorylation causes dispersion of YT521-B from YT bodies to the nucleoplasm. Whereas YT bodies are soluble in non-denaturing buffers, the phosphorylated, dispersed form is non-soluble. Non-phosphorylated YT521-B changes alternative splice site selection of the IL-4 receptor, CD44 and SRp20, but phosphorylation of c-Abl minimizes this concentration dependent effect. We propose that tyrosine phosphorylation causes sequestration of YT521-B in an insoluble nuclear form, which abolishes the ability of YT521-B to change alternative splice sites.

Alternative Splicing↗

PubMed2004

Human tra2-beta1 autoregulates its protein concentration by influencing alternative splicing of its pre-mRNA.

HTRA2-BETA1 is an SR-like protein that regulates alternative splice site selection in a concentration-dependent manner. Its proper concentration is important as several pathological states are associated with its change. We investigated the mechanism that controls the cellular HTRA2-BETA1 concentration and found it utilizes a negative feedback loop to regulate the splicing of its exon 2. TRA2-BETA1 binds to four enhancers present in exon 2, which activates its inclusion. Inclusion of exon 2 generates mRNAs that are not translated into proteins. Mutations of exon 2 enhancers demonstrate that TRA2-BETA1 binds a degenerate sequence GHVVGANR, which is found more frequently in exons than in introns. Hyperphosphorylation of TRA2-BETA1 strongly reduces its binding to RNA. Presence of the CLK2 kinase prevents the usage of exons 2 and 3, generating the htra2-beta3 mRNA. The resulting HTRA2-BETA3 protein lacks the first RS domain of HTRA2-BETA1, is expressed in several tissues and has no influence on tra2-beta splice site selection. HTRA2-BETA1 interacting proteins promote exon 2 skipping by sequestering it, which upregulates the HTRA2-BETA1 protein synthesis. We propose that the regulation of the tra2-beta pre-mRNA alternative splicing provides a robust and sensitive molecular sensor that measures the ratio between HTRA2-BETA1 and its interacting proteins.

Alternative Splicing↗

PubMed2004

ASD: the Alternative Splicing Database.

Alternative splicing is widespread in mammalian gene expression, and variant splice patterns are often specific to different stages of development, particular tissues or a disease state. There is a need to systematically collect data on alternatively spliced exons, introns and splice isoforms, and to annotate this data. The Alternative Splicing Database consortium has been addressing this need, and is committed to maintaining and developing a value-added database of alternative splice events, and of experimentally verified regulatory mechanisms that mediate splice variants. In this paper we present two of the products from this project: namely, a database of computationally delineated alternative splice events as seen in alignments of EST/cDNA sequences with genome sequences, and a database of alternatively spliced exons collected from literature. The reported splice events are from nine different organisms and are annotated for various biological features including expression states and cross-species conservation. The data are presented on our ASD web pages (http://www.ebi.ac.uk/asd).

Alternative Splicing↗

PubMed2004

p59(fyn)-mediated phosphorylation regulates the activity of the tissue-specific splicing factor rSLM-1.

The Sam68-like mammalian protein SLM-1 is a member of the STAR protein family and is related to SAM68 and SLM-2. Here, we demonstrate that rSLM-1 interacts with itself, scaffold-attachment factor B, YT521-B, SAM68, rSLM-2, SRp30c, and hnRNP G. rSLM-1 regulates splice site selection in vivo via a purine-rich enhancer. In contrast to the widely expressed SAM68 and rSLM-2 proteins, rSLM-1 is found primarily in brain and, to a much smaller degree, in testis. In the brain, rSLM-1 and rSLM-2 are predominantly expressed in different neurons. In the hippocampal formation, rSLM-1 is present only in the dentate gyrus, whereas rSLM-2 is found in the pyramidal cells of the CA1, CA3, and CA4 regions. rSLM-1, but not rSLM-2, is phosphorylated by p59(fyn). p59(fyn)-mediated phosphorylation abolishes the ability of rSLM-1 to regulate splice site selection, but has no effect on rSLM-2 activity. This suggests that rSLM-1-positive cells could respond with a change of their splicing pattern to p59(fyn) activation, whereas rSLM-2-positive cells would not be affected. Together, our data indicate that rSLM-1 is a tissue-specific splicing factor whose activity is regulated by tyrosine phosphorylation signals emanating from p59(fyn).

Adaptor Proteins, Signal Transducing↗

PubMed2004

Tau exon 10, whose missplicing causes frontotemporal dementia, is regulated by an intricate interplay of cis elements and trans factors.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. In humans, exon 10 of the gene is an alternatively spliced cassette which is adult-specific and which codes for a microtubule binding domain. Mutations that affect splicing of exon 10 have been shown to cause inherited frontotemporal dementia (FTDP). In this study, we reconstituted naturally occurring exon 10 FTDP mutants and classified their effects on its splicing. We also carried out a comprehensive survey of the influence of splicing regulators on exon 10 inclusion and tentatively identified the site of action for several of these factors. Lastly, we identified the domains of regulators SWAP and hnRNPG, which are required for regulation of exon 10 splicing.

Animals↗

PubMed2004

Expression of splicing factors in human ovarian cancer.

Alternative splicing represents an important nuclear mechanism in the post-transcriptional regulation of gene expression, which is frequently altered during tumorigenesis. Previously, we have described marked changes in alternative splicing of the CD44 gene in ovarian and breast cancer. In the latter one we described also a specific induction of splicing factors during tumor development. Now we have focussed our studies on the expression profiles of splicing factors, including classical SR proteins, Tra2 and YB-1 in physiological and malignant ovarian tissues by RT-PCR and Western blot analysis. We detected changed expression pattern with higher levels of phosphorylated 30 kDa SR proteins as well as relatively high concentrations of hyperphosphorylated Tra2 protein isoforms in ovarian cancer. RT-PCR analysis revealed a marked induction of SC35 and ASF/SF2 as well as mRNA levels in malignant ovarian tissue. These results suggest gene-specific alterations of expression rather than a general induction of the splicing machinery. Together with previously performed functional studies of CD44 splicing these findings implicate that altered expression profiles of SR proteins, Tra2beta and YB-1 might be responsible for the known changes of alternative CD44 splicing in ovarian cancer.

Antigens, CD↗

PubMed2003

Mutations in tau gene exon 10 associated with FTDP-17 alter the activity of an exonic splicing enhancer to interact with Tra2 beta.

Mutations in the human tau gene leading to aberrant splicing have been identified in FTDP-17, an autosomal dominant hereditary neurodegenerative disorder. Molecular mechanisms by which such mutations cause tau aberrant splicing were not understood. We characterized two mutations in exon 10 of the tau gene, N279K and Del280K. Our results revealed an exonic splicing enhancer element located in exon 10. The activity of this AG-rich splicing enhancer was altered by N279K and Del280K mutations. This exonic enhancer element interacts with human Tra2 beta protein. The interaction between Tra2 beta and the exonic splicing enhancer correlates with the activity of this enhancer element in stimulating splicing. Biochemical studies including in vitro splicing and RNA interference experiments in transfected cells support a role for Tra2 beta protein in regulating alternative splicing of human tau gene. Our results implicate the human tau gene as a target gene for the alternative splicing regulator Tra2 beta, suggesting that Tra2 beta may play a role in aberrant tau exon 10 alternative splicing and in the pathogenesis of tauopathies.

Alternative Splicing↗

PubMed2003

Differential regulation of 5' splice variants of the glutamate transporter EAAT2 in an in vivo model of chemical hypoxia induced by 3-nitropropionic acid.

Defective glutamate uptake has been implicated as a pathogenic event of neuronal damage related to cerebral ischemia and hypoxia. In several models of ischemia-hypoxia, a reduced immunoreactivity and altered RNA expression of excitatory amino acid transporter 2 (EAAT2), the major excitatory amino acid transporter, have been reported. However, the gene regulation of EAAT2 under these conditions is incompletely understood. In this study, we investigated alternative splicing of EAAT2 in an in vivo mouse model of chemical hypoxia as induced by 3-nitropropionic acid (3-NP). The neurotoxin 3-NP is an inhibitor of mitochondrial energy production. Furthermore, it is known to inhibit glutamate reuptake directly, representing at least one of the mechanisms responsible for 3-NP-induced neurodegeneration. Here we report an expression analysis of five known (mEAAT2/5UT1-5) and two novel (mEAAT2/5UT6, -7) 5' splice variants of EAAT2 using semiquantitative PCR. The RNA expression was studied at 2, 12, 24, 48, and 72 hr and 7 days after 3-NP administration. mEAAT2/5UT4 and mEAAT2/5UT5 were up-regulated in the frontal cortex and down-regulated in the hippocampus 12-72 hr after chemical hypoxia. In the cerebellum, there was an increased expression of mEAAT2/5UT4 and a down-regulation of mEAAT2/5UT5. mEAAT2/5UT3 show a different regional expression pattern, being regulated in the cerebellum only. mEAAT2/5UT1-7 encoded distinct 5' regulatory sequences, including conserved elements of translational control. It is easily conceivable that expression alterations of 5' splice variants of EAAT2 are related to glutamate transporter malfunction after chemical hypoxia. Our findings contribute to the hypothesis that RNA splicing events can serve as a molecular mechanism of posthypoxic gene regulation.

Alternative Splicing↗

PubMed2003

Emerin interacts in vitro with the splicing-associated factor, YT521-B.

Emerin is a nuclear membrane protein that interacts with lamin A/C at the nuclear envelope. Mutations in either emerin or lamin A/C cause Emery-Dreifuss muscular dystrophy (EDMD). The functions of emerin are poorly understood, but EDMD affects mainly skeletal and cardiac muscle. We used a high-stringency yeast two-hybrid method to screen a human heart cDNA library, with full-length emerin as bait. Four out of five candidate interactors identified were nuclear proteins: lamin A, splicing factor YT521-B, proteasome subunit PA28 gamma and transcription factor vav-1. Specific binding between emerin and the functional C-terminal domain of YT521-B was confirmed by pull-down assays and biomolecular interaction analysis (BIAcore). Inhibition by emerin of YT521-B-dependent splice site selection in vivo suggests that the interaction is physiologically significant. A 'bipartite' binding site for YT521-B in emerin was identified using alanine substitution or disease-associated mutations in emerin. The transcription factor GCL (germ cell-less) has previously been shown to bind to the same site. The results are consistent with an emerging view that lamins and lamina-associated proteins, like emerin, have a regulatory role, as well as a structural role in the nucleus. YT521-B joins a growing list of candidates for a role in a gene expression model of the pathogenesis of EDMD.

Binding Sites↗