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Results for “Alternative Splicing”

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PubMed1994

Developmentally regulated alternative splicing of a nematode type IV collagen gene.

A comparison of the genomic DNA sequence that encodes the Ascaris suum alpha 2(IV) collagen chain with the corresponding cDNA sequence led to the identification of a putative exon that was not expressed in the cDNA. The identification of this putative exon raised the possibility that transcripts of the alpha 2(IV) gene may undergo alternative splicing. We have used a reverse transcriptase-polymerase chain reaction assay to establish that such alternative splicing does indeed occur. Our results show that the A. suum alpha 2(IV) collagen gene produces at least two similar, but not identical, transcripts via the selection of two alternative exons. Furthermore, this alternative splicing appears to be developmentally regulated, suggesting that alternative splicing may be used in order to modify the properties of type IV collagen during nematode development.

Alternative Splicing↗

PubMed2000

A test of translational selection at 'silent' sites in the human genome: base composition comparisons in alternatively spliced genes.

Natural selection appears to discriminate among synonymous codons to enhance translational efficiency in a wide range of prokaryotes and eukaryotes. Codon bias is strongly related to gene expression levels in these species. In addition, between-gene variation in silent DNA divergence is inversely correlated with codon bias. However, in mammals, between-gene comparisons are complicated by distinctive nucleotide-content bias (isochores) throughout the genome. In this study, we attempted to identify translational selection by analyzing the DNA sequences of alternatively spliced genes in humans and in Drosophila melanogaster. Among codons in an alternatively spliced gene, those in constitutively expressed exons are translated more often than those in alternatively spliced exons. Thus, translational selection should act more strongly to bias codon usage and reduce silent divergence in constitutive than in alternative exons. By controlling for regional forces affecting base-composition evolution, this within-gene comparison makes it possible to detect codon selection at synonymous sites in mammals. We found that GC-ending codons are more abundant in constitutive than alternatively spliced exons in both Drosophila and humans. Contrary to our expectation, however, silent DNA divergence between mammalian species is higher in constitutive than in alternative exons.

Alternative Splicing↗

PubMed1987

Alternative splicing of chicken fibronectin in embryos and in normal and transformed cells.

To study the alternative splicing of fibronectin during embryogenesis and oncogenic transformation, we isolated cDNA clones of chicken fibronectin. The partial amino acid sequence deduced from sequencing of these clones showed that, overall, chicken fibronectin is approximately 80% identical with mammalian fibronectins. However, two of the three known regions of alternative splicing differed from this average. The V region was significantly more divergent, and RNA from embryonic chicken liver showed a pattern of V exon splicing which was distinct from that seen in human or rat fibronectins. In contrast, the EIIIB segment was very highly conserved (96%). As in mammals, this segment and another (EIIIA) were alternatively spliced in a cell-type-specific fashion. EIIIA+ and EIIIB+ species were almost absent in liver but predominated in total embryo RNA at all times from 2.5 to 11 days postfertilization. We also examined the possible contributions of fibronectin splicing and integrin receptor expression to the loss of fibronectin on oncogenic transformation. We detected little change in fibronectin splicing, other than a slight increase in representation of EIIIB+ species in fibroblasts after transformation by Rous sarcoma virus. It was also established that the overall reduction in fibronectin mRNA level observed after transformation was not accompanied by a decrease in integrin mRNA levels, indicating that fibronectin and integrin receptors are not coordinately regulated at the transcriptional level.

Amino Acid Sequence↗

PubMed2001

Tissue-specific alternate splicing of human telomerase reverse transcriptase (hTERT) influences telomere lengths during human development.

Direct genetic manipulations have shown that telomerase-mediated telomere elongation plays a key role in determining cellular replicative capacity and senescence. The mechanisms regulating the production of an active telomerase enzyme are still predominantly unknown, although roles for transcriptional control of hTERT, alternative-splicing of hTERT transcripts, and post-translational phosphorylation of hTERT protein have been advocated. Here we show that hTERT is alternatively spliced in specific patterns by different tissue types during human development. Alternative splicing often prohibits the expression of hTERT protein containing functional reverse transcriptase domains. In these instances, telomerase activity is absent, leading to shortening of telomeres. The specific pattern of hTERT mRNA variants in human development provides evidence that alternative splicing is non-random and participates in the regulation of telomerase activity.

Alternative Splicing↗

PubMed2006

CD14-dependent modulation of NF-kappaB alternative splicing in the lung after burn injury.

Nuclear factor kappa-B (NF-kappaB), a key downstream player of the LPS signaling pathway, has been shown to undergo alternative splicing in in vitro studies. In this study, we examined the effect of injury and the role of CD14 on NF-kappaB alternative splicing using a murine burn model. CD14 knockout and respective wild-type mice were sacrificed after 18% total body surface area burn. RT-PCR and subsequent sequencing analysis revealed that injury induced multiple novel splicing variants of relA, relB, and NF-kappaB2 in the lungs of CD14 knockout but not wild-type mice. These novel variants resulted either from exon skipping, alternative usage of splicing signals, or intron retention. All but one variant resulted in a frameshift leading to premature termination of translation. These splicing variants encoded for proteins that lacked the domains essential for NF-kappaB transcription factor functions. Two NF-kappaB2 variants acquired only minor changes in their C-terminus that might affect their post-translational cleavage into active isoforms. These results suggest that alternative splicing may be one of the mechanisms by which NF-kappaB activity in the lungs can be regulated after injury. Furthermore, the CD14-mediated LPS signaling pathway may play a role in the regulation of NF-kappaB alternative splicing in the lungs after injury.

Alternative Splicing↗

PubMed2004

Imaging alternative splicing in living cells.

We have developed an in vivo reporter of alternative splicing decisions that allows for the determination of FGF-R2 splicing patterns without the destruction of cells. This method has broad applications, including the study of other alternatively spliced genes in tissue culture and in whole animals, and may be useful in creating imaging markers for the study of tumor progression and metastasis. In this chapter, the authors present one example of this method using fluorescence reporters. As with any new assay, a series of experiments were performed to validate the method. This chapter documents some of these experiments.

Alternative Splicing↗

PubMed1993

cDNA structure, expression and nucleic acid-binding properties of three RNA-binding proteins in tobacco: occurrence of tissue-specific alternative splicing.

Three cDNAs encoding RNA-binding proteins were isolated from a tobacco (Nicotiana sylvestris) cDNA library. The predicted proteins (RGP-1) are homologous to each other and consist of a consensus-sequence type RNA-binding domain of 80 amino acids in the N-terminal half and a glycine-rich domain of 61-78 amino acids in the C-terminal half. Nucleic acid-binding assay using the in vitro synthesized RGP-1 protein confirmed that it is an RNA-binding protein. Based on its strong affinity for poly(G) and poly(U), the RGP-1 proteins are suggested to bind specifically to G and/or U rich sequences. All three genes are expressed in leaves, roots, flowers and cultured cells, however, the substantial amount of pre-mRNAs are accumulated especially in roots. Sequence analysis and ribonuclease protection assay indicated that significant amounts of alternatively spliced mRNAs, which are produced by differential selection of 5' splice sites, are also present in various tissues. Tissue-specific alternative splicing was found in two of the three genes. The alternatively spliced mRNAs are also detected in polysomal fractions and are suggested to produce truncated polypeptides. A possible role of this alternative splicing is discussed.

Alternative Splicing↗

PubMed1995

[Expression of CD44 alternative splicing variants in lung cancer].

Expression of isoforms of the CD44 is generated by alternative splicing of CD44 gene; CD44H: lacks all 10 alternative exons, CD44R: the alternative exons v8 to v10, CD44V: other group of variants which contains the alternative exon v6. In some tumors such as colorectal cancer, breast cancer, non-Hodgkin lymphoma, and melanoma, over-expressed CD44 isoform which contains such alternative-spliced variant exons may play a causative role in tumor metastasis. In lung cancer, however, the role of CD44 variants in tumor progression and metastasis is uncertain. In our study and reported literature, no definite correlation was observed between the expression of specific CD44 isoform and tumor progression or metastasis of lung cancer.

Alternative Splicing↗

PubMed1997

Developmentally regulated alternative splicing of mRNAs encoding N-terminal tau variants in the rat hippocampus: structural and functional implications.

Tau protein variants are axonal microtubule-associated phosphoproteins whose expression correlates with developmentally regulated neurite outgrowth. A single gene encodes multiple tau transcripts via complex alternative splicing. We studied the expression of the mRNAs encoding N-terminal variants of tau, and we showed distinct alternative splicing of exons 2 and 3 in nervous tissues of the adult rat, including the inner ear, hippocampus, cortex, striatum, brainstem, cerebellum, olfactory bulb and retina. Using the reverse transcriptase-coupled polymerase chain reaction and in situ hybridization, we then focused our developmental study on hippocampal neurons, both in vivo and in vitro, to address the developmental and spatial expression of the alternatively spliced mRNAs encoding N-terminal variants of tau. Tau mRNAs devoid of exons 2 and 3 were present throughout development, although their levels decreased in adults. Those containing exon 2 but not exon 3 were already present in the hippocampus of newborn rats and their levels increased during the first postnatal week, mainly in the pyramidal cell layer. Tau RNAs containing exons 2 and 3 appeared at the end of this period in the pyramidal cell layer and in the dentate granule cells. Exon 2-containing mRNAs seemed to be associated with cells undergoing axonal sprouting, while exon 3-containing RNAs were expressed in mature neurons that had established their connections. The timing and pattern of tau alternative splicing were maintained in cultured hippocampal neurons, suggesting that splicing processes are independent of the organized connectivity and of the environmental cues provided in vivo. Secondary structure predictions of tau variants revealed that the insertion of the exon 3-encoded domain substantially modifies the secondary structure of the N-terminal region of tau. This N-terminal heterogeneity may confer distinct regulatory roles on the tau variants during ontogeny and may contribute to plasticity in the adult rat brain.

Alternative Splicing↗

PubMed1989

Role of the branch site/3'-splice site region in adenovirus-2 E1A pre-mRNA alternative splicing: evidence for 5'- and 3'-splice site co-operation.

The adenovirus E1A gene encodes five overlapping mRNAs which are processed by alternative RNA splicing from a common pre-mRNA. To characterize cis-acting sequence elements which are of importance for the alternative 5'-splice site selection deletion and substitution mutants within the intron that is common to all E1A mRNAs were constructed. Deletion of the wild-type E1A branch site/polypyrimidine tract resulted in activation of a functionally redundant sequence located within an A/T rich sequence just upstream of the normal E1A lariat branch site. Removal of both regulatory sequences abolished in vivo splicing completely and did not lead to activation of cryptic 3'-splice sites at other locations in the E1A pre-mRNA. Furthermore we show that the sequence around the E1A branch site/3'-splice site region may have a more direct effect on the efficiency by which the alternative E1A 5'-splice sites are selected. Replacing the E1A branch site/3'-splice site region with the corresponding sequence from the second intron of the rabbit beta-globin gene or the first intron of the major late transcription unit resulted in drastic changes in E1A 5'-splice site selection. For example, with the E1A/beta-globin hybrid gene the 9S mRNA became the most abundant E1A mRNA to accumulate. This contrasts with the wild-type E1A gene in which almost undetectable levels of 9S mRNA were produced in transient expression assays. Our results strongly suggest that a cooperative interaction between 5'- and 3'-splice sites on a pre-mRNA determines the outcome of alternative splicing.

Adenoviridae↗

PubMed2001

Cis-acting elements regulate alternative splicing of exons 6A, 6B and 8 of the alpha1(XI) collagen gene and contribute to the regional diversification of collagen XI matrices.

Consecutive exons 6A, 6B, 7 and 8 that encode the variable region of the amino-terminal domain (NTD) of the col11a1 gene product undergo a complex pattern of alternative splicing that is both tissue-dependent and developmentally regulated. Expression of col11a1 is predominantly associated with cartilage where it plays a critical role in skeletal development. At least five splice-forms (6B-7-8, 6A-7-8, 7-8, 6B-7 and 7) are found in cartilage. Splice-forms containing exon 6B or 8 have distinct distributions in the long bone during development, while in non-cartilage tissues, splice-form 6A-7-8 is typically expressed. In order to study this complex and tissue-specific alternative splicing, a mini-gene that contains mouse genomic sequence from exon 5 to 11, flanking the variable region of alpha1(XI)-NTD, was constructed. The minigene was transfected into chondrocytic (RCS) and non-chondrocytic (A204) cell lines that endogenously express alpha1(XI), as well as 293 cells which do not express alpha1(XI). Alternative splicing in RCS and A204 cells reflected the appropriate cartilage and non-cartilage patterns while 293 cells produced only 6A-7-8. This suggests that 6A-7-8 is the default splicing pathway and that cell or tissue-specific trans-acting factors are required to obtain pattern of the alternative splicing of alpha1(XI) pre-mRNA observed in chondrocytes. Deletional analysis was used to identify cis-acting regions important for regulating splicing. The presence of the intact exon 7 was required to generate the full complex chondrocytic pattern of splicing. Furthermore, deletional mapping of exon 6B identified sequences required for expression of exon 6B in RCS cells and these may correspond to purine-rich (ESE) and AC-rich (ACE) exonic splicing enhancers.

Alternative Splicing↗

PubMed1995

Characterization and expression of multiple alternatively spliced transcripts of the Goodpasture antigen gene region. Goodpasture antibodies recognize recombinant proteins representing the autoantigen and one of its alternative forms.

Collagen IV, the major component of basement membranes, is composed of six distinct alpha chains (alpha 1-alpha 6). Atypically among the collagen IV genes, the exons encoding the carboxyl-terminal region of the human alpha 3(IV) chain undergo alternative splicing. This region has been designated as the Goodpasture antigen because of its reactivity in the kidney and lung with the pathogenic autoantibodies causing Goodpasture syndrome. The data presented in this report demonstrate that, in human kidney, the gene region encompassing the Goodpasture antigen generates at least six alternatively spliced transcripts predicting five distinct proteins that differ in their carboxyl-terminus and retain, except in one case, the exon that harbors the characteristic amino-terminus of the antigen. Goodpasture antibodies specifically recognize recombinant proteins representing the antigen and the alternative form that retains the amino-half of the antigen, suggesting that this moiety could be involved in the in vivo binding of the pathogenic antibodies. Furthermore, the sera of control individuals contain autoantibodies against the antigen that can be differentiated from those causing the syndrome based on their specific reactivities, suggesting that the binding of the pathogenic autoantibodies to a specific determinant likely trigger a distinct and unique cascade of events causing the disease.

Alternative Splicing↗

PubMed1993

A novel tenascin type III repeat is part of a complex of tenascin mRNA alternative splices.

Sequence analysis of two human tenascin encoding cDNA clones from a cDNA library of the U251 glioblastoma cell line revealed the presence of a novel 276 bp tenascin type III fibronectin like repeat. This alternatively spliced type III repeat designated AD1 is located between the previously identified repeats 10 and 11 and has sequence homology with human, chicken and mouse tenascin type III repeats. These results show that tenascin has at least 16 consecutive fibronectin like type III repeats. PCR amplification of random primed mRNA with specific type III repeat primers revealed a pattern of multiple alternative splices of AD1 and flanking type III repeats. The alternative splice variants were confirmed by direct sequencing. Differences were observed in the expression of the various alternative splices of tenascin mRNA between tumor and normal cells and may thus indicate differences in tenascin isoform expression and function in normal and tumor cells. PCR and Southern analysis of genomic DNA indicate that AD1 is coded by a single exon present in both human and mouse genome.

Amino Acid Sequence↗

PubMed2004

Organization and expression of thirteen alternatively spliced exons in catfish CD45 homologs.

CD45, also known as LCA, is a transmembrane protein tyrosine phosphatase encoded by the PTPRC gene. In mammals, it plays an important role in T and B cell receptor and cytokine signaling by maintaining receptor associated kinases in an active state. A prominent CD45 feature is alternative splicing of exons encoding the N-terminus, resulting in the generation of several isoforms. The expression of isoforms is tightly regulated and dependent on the developmental/activation state of the lymphocyte. Nevertheless, the significance of these multiple isoforms in mammals is poorly understood. In this study, the channel catfish CD45 homolog was sequenced and found to be similar to CD45 of other species. However, unlike mammalian CD45, it appears that up to 13 exons are used in producing multiple alternatively spliced CD45 variants in catfish cells. These 13 alternatively spliced exons variably encode for O-linked glycosylation sites. Several of the exons are identical or very similar, suggesting gene duplication of a block of four exons. As demonstrated by RT-PCR, many of the alternatively spliced forms of catfish CD45 are differentially expressed in lymphoid cell lines with B cells expressing larger isoforms than do T cells. Furthermore, immunoprecipitation experiments utilizing anti-catfish CD45 mAbs substantiated that different size CD45 isoforms are expressed at the protein level on catfish T and B cells.

Alternative Splicing↗

PubMed1994

Alternative splicing of beta-tropomyosin pre-mRNA: multiple cis-elements can contribute to the use of the 5'- and 3'-splice sites of the nonmuscle/smooth muscle exon 6.

We previously found that the splicing of exon 5 to exon 6 in the rat beta-TM gene required that exon 6 first be joined to the downstream common exon 8 (Helfman et al., Genes and Dev. 2, 1627-1638, 1988). Pre-mRNAs containing exon 5, intron 5 and exon 6 are not normally spliced in vitro. We have carried out a mutational analysis to determine which sequences in the pre-mRNA contribute to the inability of this precursor to be spliced in vitro. We found that mutations in two regions of the pre-mRNA led to activation of the 3'-splice site of exon 6, without first joining exon 6 to exon 8. First, introduction of a nine nucleotide poly U tract upstream of the 3'-splice site of exon 6 results in the splicing of exon 5 to exon 6 with as little as 35 nucleotides of exon 6. Second, introduction of a consensus 5'-splice site in exon 6 led to splicing of exon 5 to exon 6. Thus, three distinct elements can act independently to activate the use of the 3'-splice site of exon 6: (1) the sequences contained within exon 8 when joined to exon 6, (2) a poly U tract in intron 5, and (3) a consensus 5'-splice site in exon 6. Using biochemical assays, we have determined that these sequence elements interact with distinct cellular factors for 3'-splice site utilization. Although HeLa cell nuclear extracts were able to splice all three types of pre-mRNAs mentioned above, a cytoplasmic S100 fraction supplemented with SR proteins was unable to efficiently splice exon 5 to exon 6 using precursors in which exon 6 was joined to exon 8. We also studied how these elements contribute to alternative splice site selection using precursors containing the mutually exclusive, alternatively spliced cassette comprised of exons 5 through 8. Introduction of the poly U tract upstream of exon 6, and changing the 5'-splice site of exon 6 to a consensus sequence, either alone or in combination, facilitated the use of exon 6 in vitro, such that exon 6 was spliced more efficiently to exon 8. These data show that intron sequences upstream of an exon can contribute to the use of the downstream 5'-splice, and that sequences surrounding exon 6 can contribute to tissue-specific alternative splice site selection.

Alternative Splicing↗

PubMed2002

Alternatively spliced variants of the follicle-stimulating hormone receptor gene in the testis of infertile men.

OBJECTIVE: To investigate whether or not alternatively spliced variants of the FSH receptor gene occur in human testis and whether the presence of the splicing variants is associated with spermatogenic defects and serum FSH concentration in infertile men. DESIGN: A prospective case control study. SETTING: An IVF clinic and infertility laboratory at a university hospital. PATIENT(S): Forty-three infertile patients undergoing testicular biopsy. INTERVENTION(S): Total RNA was extracted from the testicular tissues and used for reverse transcriptase-polymerase chain reaction (RT-PCR). MAIN OUTCOME MEASURE(S): Expression pattern was analyzed by nested RT-PCR using primers designed to amplify a fragment of FSH receptor gene. PCR products of splicing variants were cloned and sequenced. RESULT(S): The PCR products showed three kinds of additional bands corresponding to alternatively spliced isoforms of the FSH receptor gene. Exon 9 deleted variant was detected in all patients and inclusion variant of small extra exon was detected in 64% (9/14) of the patients with normal spermatogenesis and 34% (10/29) of the patients with spermatogenic defects. The presence of inclusion variant was not significantly associated with spermatogenic defects but was associated with a low level of serum FSH. On the other hand, exon 6 deleted variant was detected in only one patient having a high level of FSH concentration (30 IU/L) and Sertoli cell only syndrome. CONCLUSION(S): We identified three different types of alternatively spliced variants of the human FSH receptor. However, it is not clear whether or not there is an association between three variants and spermatogenic defects.

Alternative Splicing↗

PubMed2003

A novel homozygous splice junction mutation in GPIIb associated with alternative splicing, nonsense-mediated decay of GPIIb-mRNA, and type II Glanzmann's thrombasthenia.

This work reports the study of a patient suffering a bleeding disorder clinically diagnosed as Glanzmann's thrombasthenia (GT). Immunoblotting and flow cytometric analysis showed a low (</= 10% of control) platelet content of GPIIb-IIIa, confirming it was indeed a type II GT. The molecular genetic analysis of the proband revealed the presence of a homozygous G188A transition in GPIIb. This mutation alters the consensus sequence of the splice donor site of intron 1 changing arginine 63 for lysine (R63K). No other mutation than [G188A]GPIIb was found in the proband and her parents after complete analysis of GPIIb and GPIIIa coding sequences, and the promoter, 3'-UTR, and intronic flanking regions of GPIIb. The GT phenotype of the proband is the result of a limited availability of GPIIb-mRNA. The etiopathogenic role of the [G188A]GPIIb mutation is supported by the following observations: (i) both parents, who are heterozygous for the [G188A]GPIIb mutation, show a marked decrease in the platelet content of GPIIb-mRNA; (ii) exontrap analysis demonstrated that the G188A mutation leads to a marked reduction in the steady-state level of GPIIb-mRNA. The reduced availability of platelet GPIIb-mRNA associated with the G188A mutation seems to be caused by either inefficient RNA splicing or a preferred utilization of alternative intronic donor sites that generate an in-frame STOP codon with the result of activation of nonsense-mediated mRNA decay, or both.

Alternative Splicing↗

PubMed1996

APP gene family. Alternative splicing generates functionally related isoforms.

The Alzheimer's beta A4-amyloid protein precursor (APP) and the APP-like proteins (APLPs) are transmembrane glycoproteins with a similar modular domain structure. APP exists in 8 isoforms generated by alternative splicing of exons 7, 8, and 15, of which the L-APP mRNAs lacking exon 15 are ubiquitously expressed in rat tissues but not in neurons. Rat APLP2, the nearest relative of APP, is similarly expressed in 4 different isoforms due to alternative splicing of inserts encoding a Kunitz protease inhibitor domain (KPI, homologous to exon 7 of APP) and a divergent region of 12 amino acids on the NH2-terminal side of the transmembrane domain (12 aa exon). KPI-APLP2 transcripts are highly expressed in neurons, in contrast to KPI-APPs, while L-APLP2 mRNA isoforms lacking the 12 aa exon are predominantly expressed in non-neuronal rat tissues, similar to L-APPs. Further examination of the divergent domains in APP and APLP2 harboring the similarly alternatively spliced APP exon 15 and the 12 aa exon of APLP2 revealed some structural similarities of the amino acid sequences and the predicted secondary structures. In both L-APLP2 and L-APP, a putative xylosyl-transferase recognition site for chondroitin sulfate glycosaminoglycan attachment is present that is interrupted in APP and APLP2 isoforms expressing APP exon 15 or the 12 aa exon of APLP2. Thus, a related function of the divergent domains and the corresponding alternatively spliced APP and APLP2 isoforms in regulation of the binding properties of the ectodomain is suggested. Additionally, beta-secretase cleavage of APP might be sterically hindered selectively in proteoglycan L-APP but not in APP lacking the proteoglycan attachment site. Neurons which have a uniquely low portion of L-APP and high content of APP might therefore be especially susceptible to beta A4-protein liberation. This could explain the selective vulnerability of neurons that is observed in Alzheimer's disease.

Alternative Splicing↗