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Results for “Alternative Splicing”

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PubMed2002

Transcriptional activators differ in their abilities to control alternative splicing.

Promoter and enhancer elements can influence alternative splicing, but the basis for this phenomenon is not well understood. Here we investigated how different transcriptional activators affect the decision between inclusion and exclusion (skipping) of the fibronectin EDI exon. A mutant of the acidic VP16 activation domain called SW6 that preferentially inhibits polymerase II (pol II) elongation caused a reduction in EDI exon skipping. Exon skipping was fully restored in the presence of the SW6 mutant by either the SV40 enhancer in cis or the human immunodeficiency virus (HIV) Tat in trans, both of which specifically stimulate pol II elongation. HIV Tat also cooperated with the Sp1 and CTF activation domains to enhance transcript elongation and EDI skipping. The extent of exon skipping correlated with the efficiency with which pol II transcripts reach the 3' end of the gene but not with the overall fold increase in transcript levels caused by different activators. The ability of activators to enhance elongation by RNA polymerase II therefore correlates with their ability to enhance exon skipping. Consistent with this observation, the elongation inhibitor dichlororibofuranosylbenzimidazole (DRB) enhanced EDI inclusion. Conversely, the histone deacetylase inhibitor trichostatin A that is thought to stimulate elongation caused a modest inhibition of EDI inclusion. Together our results support a kinetic coupling model in which the rate of transcript elongation determines the outcome of two competing splicing reactions that occur co-transcriptionally. Rapid, highly processive transcription favors EDI exon skipping, whereas slower, less processive transcription favors inclusion.

Alternative Splicing↗

PubMed1993

SnoI, a novel alternatively spliced isoform of the ski protooncogene homolog, sno.

We have cloned and sequenced a novel human isoform of sno, snoI for insertion. SnoI contains 1330 nucleotides inserted in place of 7 nucleotides of the snoN mRNA. Sno is a member of the ski protooncogene family, which has been implicated in muscle development. The two previously known sno alternatively spliced isoforms are snoN (684 amino acids), and snoA (415 amino acids); snoI encodes a truncated isoform of 399 amino acids (44,298 MW). Southern blot experiments show that snoI contains a third alternative exon from the sno gene; a single sno gene can express all three isoforms of sno by alternative splicing. All three isoforms contain the region that is most similar to the ski proto-oncogene. The relationship between snoI and snoN is analogous to that between delta fosB and fosB, where a truncated form of the fosB transcription factor is produced by alternative splicing. We find conservation of human snoI-specific sequences in several mammalian species, in monkey, dog, cow, rabbit and pig, but not in rodents, whereas the common portion of the sno gene is conserved in all vertebrate species tested. SnoN, snoA, and ski mRNAs accumulate in many human tissues including skeletal muscle; the snoI alternative mRNA accumulates more specifically in skeletal muscle. SnoI is also expressed in rhabdomyosarcoma tumor, a tumor that contains differentiated skeletal muscle. The tissue-specific alternative splicing of human snoI, an mRNA in the ski/sno gene family, and the presence of sno mRNAs in muscle are consistent with a proposed role for the sno oncogene in muscle gene regulation.

Alternative Splicing↗

PubMed2002

Alternative splicing of a beta4 subunit proline-rich motif regulates voltage-dependent gating and toxin block of Cav2.1 Ca2+ channels.

Ca2+ channel beta subunits modify alpha1 subunit gating properties through direct interactions with intracellular linker domains. In a previous report (Helton and Horne, 2002), we showed that alternative splicing of the beta4 subunit had alpha1 subunit subtype-specific effects on Ca2+ channel activation and fast inactivation. We extend these findings in the present report to include effects on slow inactivation and block by the peptide toxin omega-conotoxin (CTx)-MVIIC. N-terminal deletion and site-directed mutagenesis experiments revealed that the effects of alternative splicing on toxin block and all aspects of gating could be attributed to a proline-rich motif found within N-terminal beta4b amino acids 10-20. Interestingly, this motif is conserved within the third postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1 domain of the distantly related membrane-associated guanylate kinase homolog, PSD-95. Sequence identity of approximately 30% made possible the building of beta4a and beta4b three-dimensional structural models using PSD-95 as the target sequence. The models (1) reveal that alternative splicing of the beta4 N terminus results in dramatic differences in surface charge distribution and (2) localize the proline-rich motif of beta4b to an extended arm structure that flanks what would be the equivalent of a highly modified PSD-95 carboxylate binding loop. Northern blot analysis revealed a markedly different pattern of distribution for beta4a versus beta4b in the human CNS. Whereas beta4a is distributed throughout evolutionarily older regions of the CNS, beta4b is concentrated heavily in the forebrain. These results raise interesting questions about the functional role that alternative splicing of the beta4 subunit has played in the evolution of complex neural networks.

Alternative Splicing↗

PubMed1995

In vivo expression of alternatively spliced forms of integrin-associated protein (CD47).

Integrin-associated protein (IAP) is a 50 kDa plasma membrane protein physically and functionally associated with beta 3 integrins in a variety of cells. IAP has an extracellular immunoglobulin domain, five transmembrane domains and a short intracytoplasmic tail. IAP is recognized by anti-CD47 antibodies and is expressed on cells, such as erythrocytes and lymphocytes, which do not express beta 3 integrins. To learn more about potential functions of IAP we examined its expression in vivo. Using the polymerase chain reaction, we detected 4 alternatively splice forms of IAP which differ from each other only at their intracytoplasmic carboxy termini. These alternatively spliced forms are generated by inclusion or exclusion of three short exons within 5 kb in the genome and are highly conserved between mouse and man. There is tissue specificity of expression of the alternatively spliced forms of IAP mRNA, with bone marrow-derived cells expressing predominantly one form and neural tissue another. Using polyclonal antibodies which recognize the alternatively spliced bone marrow (form 2) and neural (form 4) forms of IAP, we found that in accord with the mRNA, form 2 protein was expressed in all tissues primarily on bone marrow-derived cells and endothelia, while form 4 was highly expressed in the brain and peripheral nervous system. The evolutionary conservation of IAP isoforms and their tissue-specific expression suggest an important role for these intracytoplasmic domains in IAP function.

Alternative Splicing↗

PubMed1998

Alternative splicing of the latency-related transcript of bovine herpesvirus 1 yields RNAs containing unique open reading frames.

The latency-related transcript (LRT) of bovine herpesvirus 1 (BHV-1) is the only abundant viral RNA detected during latency. A previous study (A. Hossain, L. M. Schang, and C. Jones, J. Virol. 69:5345-5352, 1995) concluded that splicing of polyadenylated [poly(A)+] and splicing of nonpolyadenylated [poly(A)-] LRT are different. In this study, splice junction sites of LRT were identified. In trigeminal ganglia of acutely infected calves (1, 7, or 15 days postinfection [p.i.]) or in latently infected calves (60 days p.i.), alternative splicing of poly(A)+ LRT occurred. Productive viral gene expression in trigeminal ganglia is readily detected from 2 to 7 days p.i. but not at 15 days p.i. (L. M. Schang and C. Jones, J. Virol. 71:6786-6795, 1997), suggesting that certain aspects of a lytic infection occur in neurons and that these factors influence LRT splicing. Splicing of poly(A)- LRT was also detected in transfected COS-7 cells or infected MDBK cells. DNA sequence analysis of spliced LRT cDNAs, poly(A)+ or poly(A)-, revealed nonconsensus splice signals at exon/intron and intron/exon boundaries. The GC-AG splicing signal utilized by the herpes simplex virus type 1 latency-associated transcript in latently infected mice is also used by LRT in latently infected calves. Taken together, these results led us to hypothesize that (i) poly(A)+ LRT is spliced in trigeminal ganglia by neuron-specific factors, (ii) viral or virus-induced factors participate in splicing, and (iii) alternative splicing of LRT may result in protein isoforms which have novel biological properties.

Alternative Splicing↗

PubMed2002

Organization of the human PTK7 gene encoding a receptor protein tyrosine kinase-like molecule and alternative splicing of its mRNA.

Protein tyrosine kinase-7 (PTK7) is a receptor protein tyrosine kinase (RPTK)-like molecule that contains a catalytically inactive tyrosine kinase domain. We report here the genomic structure of the human PTK7 gene by screening a BAC library and DNA sequencing. The PTK7 gene is organized into 20 exons. All of the splicing junctions followed the conserved GT/AG rule. The exon-intron structure of the PTK7 gene in the region that encodes the catalytic domain was distinct from those of other RPTKs with strong homology. The 5'-flanking sequence of the PTK7 gene contains two GC boxes that concatenate Sp1 binding motifs, but does not contain either the TATA or CAAT consensus sequence. Using a luciferase reporter assay, it was demonstrated that the 883-bp 5'-flanking sequence is functional as a promoter of the PTK7 gene. We identified four new splicing variants in testis that could be derived from alternative splicing of exons 8-10, 10, a part of 12-13, and 16. The expression patterns of the splicing variants in the hepatoma and colon cancer cells were different from those of the testis. Our findings suggest that PTK7 is evolutionarily distinct from other RPTKs, and that the alternative splicing of PTK7 mRNA may contribute to its diverse function in cell signaling.

5' Flanking Region↗

PubMed2003

Splicing factor SRp30c interaction with Y-box protein-1 confers nuclear YB-1 shuttling and alternative splice site selection.

The multifunctional DNA- and RNA-associated Y-box protein 1 (YB-1) specifically binds to splicing recognition motifs and regulates alternative splice site selection. Here, we identify the arginine/serine-rich SRp30c protein as an interacting protein of YB-1 by performing a two-hybrid screen against a human mesangial cell cDNA library. Co-immunoprecipitation studies confirm a direct interaction of tagged proteins YB-1 and SRp30c in the absence of RNA via two independent protein domains of YB-1. A high affinity interaction is conferred through the N-terminal region. We show that the subcellular YB-1 localization is dependent on the cellular SRp30c content. In proliferating cells, YB-1 localizes to the cytoplasm, whereas FLAG-SRp30c protein is detected in the nucleus. After overexpression of YB-1 and FLAG-SRp30c, both proteins are co-localized in the nucleus, and this requires the N-terminal region of YB-1. Heat shock treatment of cells, a condition under which SRp30c accumulates in stress-induced Sam68 nuclear bodies, abrogates the co-localization and YB-1 shuttles back to the cytoplasm. Finally, the functional relevance of the YB-1/SRp30c interaction for in vivo splicing is demonstrated in the E1A minigene model system. Here, changes in splice site selection are detected, that is, overexpression of YB-1 is accompanied by preferential 5' splicing site selection and formation of the 12 S isoform.

Adenovirus E1A Proteins↗

PubMed2004

Novel down-regulatory mechanism of the surface expression of the vasopressin V2 receptor by an alternative splice receptor variant.

In rat kidney, two alternatively spliced transcripts are generated from the V2 vasopressin receptor gene. The large transcript (1.2 kb) encodes the canonical V2 receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V2 receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V2 receptor (here called the V2a receptor). Cells expressing the V2b splice variant do not exhibit binding to 3H-labeled vasopressin. Interestingly, we found that expression of the splice variant V2b down-regulates the surface expression of the V2a receptor, most likely via the formation of V2a.V2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V2a receptor and the V2b splice variant. The V2b splice variant would then be acting as a dominant negative. The effect of the V2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled interleukin-8 receptor (CXCR1). Furthermore, the sequence encompassing residues 242-339, corresponding to the C-terminal domain of the V2b splice variant, also down-regulates the surface expression of the V2a receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V2b, which could retain the wild-type V2a receptor inside the cell via the formation of V2a.V2b heterodimers.

Alternative Splicing↗

PubMed2003

Regulation of alternative splicing by SRrp86 and its interacting proteins.

SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.

Adenosine Triphosphatases↗

PubMedDate not supplied

Alternative splicing of mRNA of mouse interleukin-4 and interleukin-6.

Interleukin-4 and interleukin-6 are multifunctional regulatory proteins, which participate both in haemopoiesis and in immunopoiesis. The alternative splicing of these interleukins in humans is known to proceed in a tissue-specific manner. Additionally, changes in splicing can also be dependent on tissue pathology. In this work, we report on the presence of alternatively spliced mRNA (IL-4delta2mRNA), lacking exon 2, in mouse bone marrow and spleen cells. We find that in unstimulated cells IL-4mRNA levels strongly dominate over IL-4delta2mRNA levels. Both increase in response to stimulation, with the concentration of the alternative variant rising earlier and faster than that of the full-length variant. In all other tissues studied dominance of IL-4delta2mRNA over the full-length variant was not observed. In addition, we find expression of three forms of IL-6 mRNA: the full-length IL-6 mRNA, IL-6Delta3 mRNA, and IL-6Delta5 mRNA in the second and third trimester placenta tissue and in the spleen of mice immunized with a high dose of sheep erythrocytes. It is anticipated that translation of these mRNA variants can generate proteins capable of binding to some subunits of the IL-6 receptor, thus possessing effector function. Alternative splicing is discussed as a source of cytokines with new regulatory properties.

Alternative Splicing↗

PubMed1995

The pre-mRNA of nuclear respiratory factor 1, a regulator of mitochondrial biogenesis, is alternatively spliced in human tissues and cell lines.

Nuclear respiratory factor 1 (nrf-1) is a transcriptional activator that is most probably essential in the regulation of mitochondrial biogenesis. In studies of the expression of the NRF-1 gene in cultured human fibroblasts, using RT-PCR, we identified two distinct transcripts, one of which contained an in-frame deletion of 198 bp. Analysis of genomic DNA by sequencing, showed that the shorter mRNA is the result of alternative splicing (exon skipping). The shorter transcript will result in an isoform of the protein that lacks the carboxy-terminal part of the DNA binding domain, which might influence transcriptional activation by normal nrf-1. The alternatively spliced transcript was also present in other human cell lines and in several human tissues. A quantitative PCR analysis showed that the percentages of the alternatively spliced transcript ranged from 3 to 17%. Differences in the percentage of alternatively spliced NRF-1 pre-mRNA may influence mitochondrial biogenesis under variable physiological conditions and could play a role in distinct mitochondrial diseases.

Alternative Splicing↗

PubMed2004

A novel CD45 transcript involving the alternative splicing of a new exon is widely distributed in lymphoid tissues.

Protein tyrosine phosphatase CD45 is a key player in T-cell receptor signaling and lymphocyte development. Differential expression of multiple CD45 isoforms resulting from the alternative splicing of exons A, B, and C, which encode part of the extracellular domain, is an important feature of CD45 expression. We report a novel isoform that results from the alternative splicing of a previously undiscovered exon between the constitutively spliced exon 3 and the alternatively spliced exon A. This 123-bpâ&#x0080:#x0093:long exon encodes 41 amino acids and is unlikely to undergo te extensive glycosylation seen for the regions encoded by exons A, B, and C. Reverse transcriptase polymerase chain reaction demonstrates that this isoform is expressed in human peripheral blood mononuclear cells and cell lines of lymphoid origin, but with a clearly different pattern to that of the isoforms caused by exons A, B, and C, implying a different regulatory mechanism.

Alternative Splicing↗

PubMed2006

Unbalanced alternative splicing and its significance in cancer.

Alternative pre-mRNA splicing leads to distinct products of gene expression in development and disease. Antagonistic splice variants of genes involved in differentiation, apoptosis, invasion and metastasis often exist in a delicate equilibrium that is found to be perturbed in tumours. In several recent examples, splice variants that are overexpressed in cancer are expressed as hyper-oncogenic proteins, which often correlate with poor prognosis, thus suggesting improved diagnosis and follow up treatment. Global gene expression technologies are just beginning to decipher the interplay between alternatively spliced isoforms and protein-splicing factors that will lead to identification of the mutations in these trans-acting factors responsible for pathogenic alternative splicing in cancer.

Alternative Splicing↗

PubMed2006

SRp54 (SFRS11), a regulator for tau exon 10 alternative splicing identified by an expression cloning strategy.

The tau gene encodes a microtubule-associated protein that is critical for neuronal survival and function. Splicing defects in the human tau gene lead to frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), an autosomal dominant neurodegenerative disorder. Genetic mutations associated with FTDP-17 often affect tau exon 10 alternative splicing. To investigate mechanisms regulating tau exon 10 alternative splicing, we have developed a green fluorescent protein reporter for tau exon 10 skipping and an expression cloning strategy to identify splicing regulators. A role for SRp54 (also named SFRS11) as a tau exon 10 splicing repressor has been uncovered using this strategy. The overexpression of SRp54 suppresses tau exon 10 inclusion. RNA interference-mediated knock-down of SRp54 increases exon 10 inclusion. SRp54 interacts with a purine-rich element in exon 10 and antagonizes Tra2beta, an SR-domain-containing protein that enhances exon 10 inclusion. Deletion of this exonic element eliminates the activity of SRp54 in suppressing exon 10 inclusion. Our data support a role of SRp54 in regulating tau exon 10 splicing. These experiments also establish a generally useful approach for identifying trans-acting regulators of alternative splicing by expression cloning.

Alternative Splicing↗

PubMed2001

Bidirectional regulation of neurite elaboration by alternatively spliced metabotropic glutamate receptor 5 (mGluR5) isoforms.

Alternative splicing in the mGluR5 gene generates two different receptor isoforms, of which expression is developmentally regulated. However, little is known about the functional significance of mGluR5 splice variants. We have examined the functional coupling, subcellular targeting, and effect on neuronal differentiation of epitope-tagged mGluR5 isoforms by expression in neuroblastoma NG108-15 cells. We found that both mGluR5 splice variants give rise to comparable [Ca2+]i transients and have similar pharmacological profile. Tagged receptors were shown by immunofluorescence to be inserted in the plasma membrane. In undifferentiated cells the subcellular localization of the two mGluR5 isoforms was partially segregated, whereas in differentiated cells the labeling largely redistributed to the newly formed neurites. Interestingly, we demonstrate that mGluR5 splice variants dramatically influence the formation and maturation of neurites; mGluR5a hinders the acquisition of mature neuronal traits and mGluR5b fosters the elaboration and extension of neurites. These effects are partly inhibited by MPEP.

Alternative Splicing↗

PubMed2002

Computational analysis of alternative splicing using EST tissue information.

Expressed sequence tags (ESTs) from normal and tumor tissues have been deposited in public databases. These ESTs and all mRNA sequences were aligned with the human genome sequence using LEADS, Compugen's alternative splicing modeling platform. We developed a novel computational approach to analyze tissue information of aligned ESTs in order to identify cancer-specific alternative splicing and gene segments highly expressed in particular cancers. Several genes, including one encoding a possible pre-mRNA splicing factor, displayed cancer-specific alternative splicing. In addition, multiple candidate gene segments highly expressed in colon cancers were identified.

Alternative Splicing↗

PubMed2001

Alternative splicing of fibroblast growth factor receptors in human prostate cancer.

BACKGROUND: Fibroblast growth factors (FGFs) are known to play an important role in the growth of normal prostatic epithelial cells and may promote proliferation of neoplastic prostatic epithelial cells via autocrine or paracrine mechanisms. The affinity of FGFs for FGF receptors 1-3 is critically dependent on an alternative splicing event involving the coding region for the carboxy terminal portion of the third extracellular immunoglobulin-like domain that leads to two different isoforms of each receptor (IIIb and IIIc). We therefore sought to determine whether changes in alternative splicing of FGF receptors occur in human prostate cancer. METHODS: RNAs from normal prostate and clinically localized or metastatic prostate cancers were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) followed by digestion of products with restriction enzymes specific for each FGF receptor isoform and quantitation of the relative amounts of each isoform after electrophoresis. For FGFR-2, this was correlated with immunohistochemistry to determine the localization of the protein product. RESULTS: FGFR-1 is expressed exclusively as the IIIc isoform in prostate cancer while FGFR-3 is expressed predominantly as the IIIb isoform, similar to the expression pattern in normal prostatic epithelial cells. In contrast, there was variable expression of the FGFR-2 IIIb and IIIc isoforms. In the majority of cases the FGFR-2 IIIb isoform was the predominant or exclusive isoform expressed, similar to normal epithelial cells, but in a subset of cases the IIIc isoform was increased, indicating a change in alternative splicing of FGFR-2 in some cases. CONCLUSIONS: In most cases of prostate cancer there are no changes in alternative splicing of FGF receptors, but in a subgroup there is increased expression of the FGFR-2 IIIc isoform. Given that the affinity of FGFs is highly dependent on the isoform of FGF receptor expressed, this information is critical in understanding the effects of FGFs on prostate cancer cells in vivo.

Alternative Splicing↗

PubMed2005

Single-nucleotide polymorphisms in NAGNAG acceptors are highly predictive for variations of alternative splicing.

Aberrant or modified splicing patterns of genes are causative for many human diseases. Therefore, the identification of genetic variations that cause changes in the splicing pattern of a gene is important. Elsewhere, we described the widespread occurrence of alternative splicing at NAGNAG acceptors. Here, we report a genomewide screen for single-nucleotide polymorphisms (SNPs) that affect such tandem acceptors. From 121 SNPs identified, we extracted 64 SNPs that most likely affect alternative NAGNAG splicing. We demonstrate that the NAGNAG motif is necessary and sufficient for this type of alternative splicing. The evolutionarily young NAGNAG alleles, as determined by the comparison with the chimpanzee genome, exhibit the same biases toward intron phase 1 and single-amino acid insertion/deletions that were already observed for all human NAGNAG acceptors. Since 28% of the NAGNAG SNPs occur in known disease genes, they represent preferable candidates for a more-detailed functional analysis, especially since the splice relevance for some of the coding SNPs is overlooked. Against the background of a general lack of methods for identifying splice-relevant SNPs, the presented approach is highly effective in the prediction of polymorphisms that are causal for variations in alternative splicing.

Alleles↗