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Results for “splicing variant”

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PubMed1997

Localization of leptin receptor mRNA splice variants in murine peripheral tissues by RT-PCR and in situ hybridization.

Expression of leptin receptor splice variants, including the long form variant (Ob-Rb), has been examined in murine peripheral tissues. RT-PCR indicates that the leptin receptor, Ob-R, and in particular the Ob-Ra splice variant are expressed in a wide range of tissues. Expression of the Ob-R receptor was localized by in situ hybridization to specific sites in the spleen, testes, kidney, liver, lung, and adrenal. However, the long form leptin receptor, Ob-Rb, was only expressed at significant levels in the medulla of the adrenal and the inner zone of the medulla of the kidney. The specific sites of expression of different splice variants within peripheral tissues has important implications with regard to the function of leptin.

Animals↗

PubMed2025

Utility of genome sequencing and group-enrichment to support splice variant interpretation in Marfan syndrome.

PURPOSE: To quantify the impact of noncanonical FBN1 splice site variants in undiagnosed Marfan syndrome (MFS), a connective tissue disorder associated with skeletal abnormalities and familial thoracic aortic aneurysm disease (FTAAD). METHODS: A systematic analysis of ultrarare FBN1 variants was performed using genome sequencing data from the 100,000 Genomes Project. Variants were annotated with SpliceAI and the significance of enrichment among individuals with FTAAD was assessed using Fisher's exact test. Experimental validation used RNA sequencing, reverse transcriptase polymerase chain reaction, minigene constructs, and replication analysis was with data from UK Biobank. RESULTS: Using aggregate data for 78,195 individuals, we identified 13,864 singleton single-nucleotide variants in FBN1 of which 21 were predicted to affect splicing (SpliceAI > 0.5). Incidence of candidate splice variants in individuals recruited with FTAAD (9/703) was significantly elevated compared with that seen in non-FTAAD participants (12/77,492; odds ratio = 84, P = 9.7 × 10-14). Additional analysis uncovered a further 14 families harboring 11 different FBN1 splice variants. A total of 20 candidate splice variants in 23 families were identified, of which 70% lay beyond the ±8 splice regions. RNA testing confirmed the predicted splice aberration in 16 of 20 and for 9 of 20, pseudoexonization was the likely splicing anomaly. CONCLUSION: Our findings indicate that noncanonical splice variants may account for approximately 3% of families with undiagnosed FTAAD, highlighting the importance of incorporating analysis of introns and confirmatory RNA testing into genetic testing for Marfan syndrome.

Humans↗

PubMedDate not supplied

Splice variants of the OB receptor gene are differentially expressed in brain and peripheral tissues of mice.

A high affinity receptor for OB protein was recently cloned from the choroid plexus of mice. At least six alternatively spliced forms of the OB receptor (OB-R) gene have been described, all of which encode proteins containing the OB-R extracellular domain. One splice variant encodes a receptor with a long intracellular domain, OB-RL, that has been implicated in OB-R signaling. Here, we have used in situ hybridization to examine the localization of OB-R splice variants in brain and peripheral tissues of adult and newborn mice. Using a probe hybridizing with all known splice variants, we confirmed that OB-R mRNA was widely distributed in the adult tissues. In the CNS, choroid plexus was the major site of expression. We now demonstrate that OB-R mRNA is expressed in peripheral tissues; primarily associated with connective tissues. In addition, OB-R mRNA was detected at higher levels in peripheral tissues of newborn mice than in adult mice. With a probe specific for OB-RL, we confirmed that high mRNA expression was detected in hypothalamic nuclei, while low levels were observed in choroid plexus. We now report that in peripheral tissues of adult mice, OB-RL mRNA expression was either very low or undetectable. In newborn mice, the pattern of OB-RL message expression in the CNS was similar to that of adult mice, while bone was the site of highest OB-RL message expression in the peripheral tissue. These data suggest different biological roles for OB-R splice variants encoding the short and long forms of OB-R. The localization of OB-RL to hypothalamic nuclei supports the idea that OB-RL is the brain receptor that mediates OB protein signaling and actions. In addition, the expression of OB-R message in newborn mice also suggests a biological role of OB-R during development in mice.

Alternative Splicing↗

PubMed1995

Translation in vivo of 5' untranslated-region splice variants of human surfactant protein-A.

Transcripts of human SP-A genes, SP-A1 and SP-A2, undergo alternative splicing of 5' untranslated-region exons. We reverse-transcribed and amplified free cytoplasmic and polysome-bound RNA and showed that (a) all splice variants of both genes are translated in vivo, (b) the relative translatability of splice variants can differ among individuals, and (c) the relative levels of different SP-A splice variants differ among individuals.

Base Sequence↗

PubMed1998

CD44 splice variant expression in clear cell carcinoma of the ovary.

OBJECTIVE: CD44 is a surface glycoprotein widely distributed among different tissues. Malignant tumors may show a more complex pattern of CD44 expression, indicating a loss of splice control. The aim of our study is to investigate the expression of CD44 splice variants (CD44v) and its metastatic potential in clear cell carcinoma of the ovary. METHODS: Twenty-two cases of clear cell carcinoma of the ovary were evaluated for CD44 standard form (CD44s) and splice variants: -4v, -6v, and -9v expression by immunocytochemistry. RESULTS: Twenty-one primary ovarian tumors and 23 metastatic sites were available for evaluation. Eighteen of 21 (86%) of ovarian sections studied expressed CD44s; 15/21 (71%) expressed CD44-4v; 14/21 (67%) expressed CD44-6v; and 12/21 (57%) expressed CD44-9v. Of 23 metastatic sites evaluated, 87% expressed CD44s. In contrast, only 5 (22%) metastases had CD44-4v and CD44-6v expression and 8 (35%) had CD44-9v immunoreactivity. None of 10 normal contralateral ovaries expressed CD44s or any splice variants. In 2 cases we had tumor available from the primary surgery, and subsequent recurrences. Both recurrences showed decreased expression of CD44-4v and CD44-6v. CONCLUSIONS: Clear cell carcinoma of the ovary shows an abnormal pattern of CD44s expression and mRNA splicing when compared to the contralateral normal ovary in the same patient. Metastases of clear cell carcinoma show a downregulation in expression of some splice variants. Furthermore, we have data that suggest that as the tumors recur, CD44s and its isoforms are downregulated. Our results suggest that alternative mRNA splicing of CD44 may be important in the development of metastases from clear cell carcinoma of the ovary.

Adenocarcinoma, Clear Cell↗

PubMed2002

Regulation of cAMP-specific phosphodiesterases type 4B and 4D (PDE4) splice variants by cAMP signaling in primary cortical neurons.

This study examined the regulation of all known phosphodiesterase (PDE) type PDE4A, PDE4B and PDE4D splice variants in cortical neurons by cAMP signaling. Treatment with dibutyryl-cAMP (db-cAMP) caused the induction of two of the known splice variants, PDE4B2 and PDE4D1/PDE4D2. Although the splice variants PDE4A1, PDE4A5/PDE4A10, PDE4B3, PDE4B1, PDE4D3 and PDE4D4 were present in cortical neurons, their mRNA was not regulated at the transcriptional level by db-cAMP. To assess the increase in PDE4B2 and PDE4D1/D2 mRNA expression, the promoters containing these genes were characterized. Transcription from both promoters was stimulated by db-cAMP. Because chronic antidepressant treatment increases PDE4B, and not PDE4D, mRNA expression, we focused on the regulation of the PDE4B2 promoter by cAMP and CREB. Dominant negative mutants of CREB suppressed PDE4B2 promoter activity and a constitutively active form of CREB robustly stimulated it. These data demonstrate that in cortical neurons, a short PDE4B2 intronic promoter is regulated by CREB, confers cAMP responsitivity and directs PDE4B2 mRNA and protein expression.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Molecular cloning and tissue distribution of an alternatively spliced variant of an A-type K+ channel alpha-subunit, Kv4.3 in the rat.

We describe here (1) the heterogeneous expression of Ca2+-independent transient (A-type) K+ channel alpha-subunits (Kv1.4, Kv3.3, Kv3.4, Kv4.2 and Kv4.3) in rat smooth muscle, heart and brain, (2) the molecular cloning and tissue distribution of a novel alternatively spliced variant of an A-type K+ channel alpha-subunit, Kv4.3, and (3) the functional expression of A-type K+ channels in HEK293 cells by the transfection with the novel splice variant of Kv4.3. A cDNA encoding this splice variant was identified from rat vas deferens by RT-PCR cloning. This cDNA clone contains a 1965 bp open reading frame that encodes for a protein of 655 amino acids. It has a 19 amino acid insertion in comparison with Kv4.3 previously reported in rat brain. RT-PCR analyses showed that the mRNAs of this longer variant are abundantly expressed in a number of smooth muscles of the rat, and that the mRNAs of the previously reported clones are absent. The longer splice variant is very weakly expressed in brain, but is the major product in heart.

4-Aminopyridine↗

PubMed2004

Sexual dimorphic expression pattern of a splice variant of zebrafish vasa during gonadal development.

In Drosophila, the RNA helicase VASA (VAS) is required for both germ line formation and oocyte differentiation. While the murine VAS homologue is required for spermatogenesis, it is dispensable for germ line formation. The molecular basis for this apparently dual role of VAS in germ line ontogeny is, however, unclear. Recent evidence indicates that fish, like flies, employs VAS both in early and late stages of the germ line development and that there is a sex-linked differential expression of splice variants. We show here that the longer of two splice variants of zebrafish vas is transiently downregulated in the germ line around the time when the germ cells reach the developing gonad. Using transgenic vas::EGFP fish lines, which allow us to distinguish between male and female individuals, we show that the long splice variant reappears in both sexes at around day 25 and is subsequently downregulated during male gonadal development. Our data further suggest that there is a switch from maternal to zygotic expression of the long splice variant of vas as sexual dimorphic development commences.

Alternative Splicing↗

PubMed2000

Glutamate transporter EAAT2 splice variants occur not only in ALS, but also in AD and controls.

OBJECTIVE: To ascertain the specificity of alternatively spliced mRNA variants of the astroglial glutamate transporter EAAT2 for ALS. BACKGROUND: An important hypothesis for ALS pathogenesis is that motor neuron injury may result from chronically elevated glutamate levels in the CNS. Supporting this idea are reports of decreased glutamate transport in ALS. This in turn has recently been suggested to be due to the presence of aberrant mRNA splice variants for EAAT2 in ALS. METHODS: Postmortem human brain tissue was obtained from different brain regions of patients with ALS, normal controls (NC), and patients with AD and Lewy body dementia (LB)-neurodegenerative diseases in which motor neurons are unaffected. Brain RNA was analyzed for EAAT2 isoforms using reverse transcription PCR and cDNA cloning/sequencing methods. RESULTS: Splice variants lacking exons 7 or 9 were present in ALS brain, as previously reported, but were also present in brains from NC, AD, and LB patients. PCR product sequence analyses from non-ALS brain show variant splicing identical to that reported for ALS. Quantitative PCR analysis shows that these isoforms may be somewhat more abundant in ALS than AD, LB, and NC brains. CONCLUSIONS: EAAT2 mRNA splice variants are found in the brains of NC and AD patients, as in ALS. The authors cannot exclude the possibility that quantitative changes in variant EAAT2 isoforms might relate directly, or indirectly, to ALS pathology. However, the qualitative presence of these "abnormal" EAAT2 splice variants does not appear to be sufficient to explain motor neuron degeneration in ALS.

Alternative Splicing↗

PubMed1995

Characterization of two splice variants of metastasis-associated human mts1 gene.

The mts1 gene is one of the genes specifically expressed in mouse metastatic tumors and tumor cell lines. In this paper, we present data on cloning and sequencing of two variants of human mts1 cDNAs (hu-mts1 and hu-mts1 (var)), as well as of the corresponding region in the human genome. Comparison of the genomic sequence with the sequence of the mts1 cDNAs demonstrates presence of two alternatively spliced variants of the mts1 in the human osteosarcoma cell line (OHS). The alternative splicing occurs within the 5'-untranslated region (UTR) of human mts1 pre-mRNA. Both splice variants, hu-mts1 and hu-mts1 (var), retain similar stability in the cells, contain one open reading frame coding for the MTS1 protein and differ only slightly in their translational capacity. The splice variants demonstrate dramatic variations in the level of expression in different human tissues and in human tumor cell lines. Although we have not revealed substantial differences in the mode of action of the two splice variants in the cells, the observed tissue specificity of expression supports the notion that it plays an important role in determining the activity of mts1 in different tissues.

Alternative Splicing↗

PubMed1999

Identification of a splice variant of neutrophil collagenase (MMP-8).

We have identified a splice variant of human neutrophil collagenase (MMP-8) transcript (MMP-8alt) that has a 91 bp insertion between codons for amino acid residues 34 and 35 of MMP-8 cDNA. This splice variant encodes an open reading frame for a 444 residue protein, lacking a secretory signal sequence. Our data suggested that, as opposed to the original MMP-8, the translation product of MMP-8alt is not a secreted protein; nevertheless, it is enzymatically active. Further studies aimed at identifying the physiological substrates of MMP-8alt protein may lead to uncover novel roles it plays in cellular physiology.

Alternative Splicing↗

PubMed2000

Distinct expression of splice variants of neuronal nitric oxide synthase in the human gastrointestinal tract.

BACKGROUND & AIMS: Changes of neuronal nitric oxide synthase (nNOS) expression have been linked to several human gastrointestinal disorders such as achalasia, diabetic gastroparesis, and hypertrophic pyloric stenosis. They could be caused by differential transcriptional control or alternative splicing generating different nNOS proteins. The aims of this study were to characterize 5'-splice variants, promoter usage, and site-specific expression of nNOS in the human gastrointestinal tract. METHODS: 5'-Splice variants were characterized by immunoblotting, reverse-transcription polymerase chain reaction, 5'-rapid amplification of complementary DNA ends, and Southern blotting. Genomic analysis was performed by rapid amplification of genomic ends, followed by reporter gene assays. RESULTS: Six different 5'-splice variants of nNOS-messenger RNA were identified showing specific expressions at various sites of the human gastrointestinal tract. Three variants encode for nNOSalpha, which has a specific N-terminal PDZ/GLGF domain and interaction sites for regulatory proteins. Two variants encode for nNOSbeta and 1 for nNOSgamma, which both lack the protein-binding domains of nNOSalpha. In addition to 2 known first exons, a novel first exon of human nNOS with a separate functionally active downstream promoter and multiple binding sites for transcription factors was identified and characterized. CONCLUSIONS: Six 5'-mRNA splice variants of nNOS encoding 3 different nNOS proteins are expressed in the human gut. The differential expression of these proteins could be implicated in different biological functions.

Alternative Splicing↗

PubMed2001

Multiple splicing variants of two new human ATP-binding cassette transporters, ABCC11 and ABCC12.

Two new human ABC transporters, ABCC11 and ABCC12, were cloned from a cDNA library of human adult liver. ABCC11 and ABCC12 genes consist of 30 and 29 exons, respectively, and they are tandemly located in a tail-to-head orientation on human chromosome 16q12.1. The predicted amino acid sequences of both gene products show a high similarity with ABCC5. The transcripts of ABCC11 and ABCC12 genes were detected by PCR in various adult human tissues, including liver, lung, and kidney, and also in several fetal tissues. By searching cDNA libraries from various human tissues, we have identified alternative splicing variants of ABCC11 and ABCC12 genes at significantly high frequencies. One splice variant lacking the exon 28 corresponded to about 25% of total ABCC11 gene transcripts. Furthermore, four splicing variants encoding putatively short peptides were predominant in ABCC12 gene transcripts. Those splicing variants may represent diverse biological functions of these ABC transporter genes.

ATP-Binding Cassette Transporters↗

PubMed2006

MUC1 splice variants in human ocular surface tissues: possible differences between dry eye patients and normal controls.

Mucins are highly glycosylated proteins that are vital to the maintenance of healthy epithelial surfaces including the ocular surface. Mucins act as lubricants, protectants, and mediators of signal transduction. The majority of the O-glycosylation sites on the transmembrane mucin MUC1 are found in a highly polymorphic core region containing a variable number of tandem repeats (VNTR). MUC1 alleles can be divided into size classes that contain small (30-45) or large (60-90) numbers of repeats. Although at least 12 splice variants of MUC1 have been found in other tissues, no splice variants have been reported in human ocular surface tissues. We have used RT-PCR to identify MUC1 splice variants that were then confirmed by sequencing. We here report the presence in some samples of human cornea, conjunctiva, and lacrimal gland of MUC1/B which features canonical splicing between exons 1 and 2 and MUC1/A, a transcript that retains 27bp from the 3' end of intron 1 and is predicted to add 9 amino acids to the MUC1 sequence upstream of the tandem repeat region. Cornea and conjunctiva both contain the MUC1/SEC splice variant that lacks the transmembrane domain and, therefore, results in a soluble, secreted form of MUC1. Cornea and conjunctiva also contain MUC1/Y and MUC1/Z(X) variants that lack the tandem repeat region. Cornea, conjunctiva, and lacrimal gland also contain a previously undescribed MUC1 variant transcript, termed MUC1/YI, that retains 99bp from the 5' end and 27bp from the 3' end of the first intron, resulting in a frame shift and premature stop codon. This transcript is predicted to produce a novel 27 amino acid peptide after signal peptidase cleavage. Analysis of brush cytology samples revealed that the percentage of dry eye patients expressing the MUC1/A variant in the conjunctival epithelium is lower than in normal control donors. Western blotting confirmed that MUC1/A is associated with alleles containing the large size class of tandem repeats. Therefore, we propose that one factor in susceptibility to dry eye disease may be the lengths of the MUC1 VNTR in conjunctival epithelium based on the rationale that longer VNTR provide better lubrication and greater protection of the ocular surface against inflammation.

Alternative Splicing↗

PubMed1997

Multiple splice variants of phosphodiesterase PDE4C cloned from human lung and testis.

Four closely related cyclic-nucleotide specific phosphodiesterase (PDE4) genes have been identified in both humans and rats: PDE4A, 4B, 4C and 4D. We have now cloned cDNAs for multiple splice variants of human PDE4C. Two splice variants, PDE4C-791 and PDE4C-426, were isolated from a fetal lung library. The longest open reading frame (ORF) of 791 amino acids (aa) is encoded by PDE4C-791, which is similar to a recently described cDNA [Engels, P., Sullivan, M., Muller, T. and Lubbert, H. FEBS Lett. 358 (1995) 305-10], except that an alternative 5'-end sequence upstream of the first methionine extends the PDE4C-791 ORF by 79 aa. The PDE4C-426 variant contains 3 insertions that are located 5' to the catalytic domain and encode several in-frame stop codons. The predicted 426 aa protein initiates at a methionine 365 aa within PDE4C-791. A baculovirus clone starting at this methionine expressed an enzymatically active protein. Two additional splice variants, PDE4C-delta54 and PDE4C-delta109, were found in testis mRNA. PDE4C-delta54 contained a novel 5'-end region and a deletion of 162 nt; the predicted protein deletes 54 aa from the amino-terminal region. The PDE4C-delta54 protein produced in baculovirus-infected cells was enzymatically active and sensitive to PDE4-specific inhibitors. The PDE4C-delta109 protein is similar to PDE4C-delta54 but has an additional 55 aa deleted in the catalytic domain; it lacked enzymatic activity. Analysis of uncloned total mRNA from 4 tissue sources by polymerase chain reaction (PCR) confirmed the presence of mRNAs with the two deletions and three insertions that we observed in cDNA clones. The PDE4C-delta54 variant was found only in testis and the 5'-extended region of PDE4C-791 was seen only in lung and the melanoma cell line G361. Hence, tissue-specific expression of various PDE4C isoforms should be considered in understanding how these gene products modulate cellular responses to cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1991

CD44 splice variants confer metastatic behavior in rats: homologous sequences are expressed in human tumor cell lines.

One of several splice variants of CD44 expressed in metastasizing cell lines of rat tumors has been shown to confer metastatic potential to the non-metastatic variant of a rat pancreatic carcinoma line (U. Günthert et al., Cell, 65: 13-24, 1991). The variant-specific rat CD44 sequences were used to detect RNA expression in human cell lines: in carcinoma lines from lung, breast and colon; and in keratinocyte lines. By polymerase chain reaction amplification, complementary DNAs encoding human homologues were isolated and sequenced. The largest splice variant has been found in a large cell lung carcinoma line and in keratinocyte cell lines. It carries at least 5 additional domains (exons) encoding a total of 338 amino acids in the membrane-proximal extracellular region of the standard CD44. Various alternative splice products have been detected in other human tumor cell lines. The distribution of CD44 splice variants is consistent with the speculation that they fulfill functions in only a few restricted differentiation pathways and that in tumor cells these pathways have been reactivated.

Amino Acid Sequence↗

PubMed1998

Cloning and functional studies of splice variants of the alpha-subunit of the amiloride-sensitive Na+ channel.

The alpha-subunit of the amiloride-sensitive epithelial Na+ channel (alpha ENaC) is critical in forming an ion conductive pore in the membrane. We have identified the wild-type and three splice variants of the human alpha ENaC (h alpha ENaC) from the human lung cell line H441, using RT-PCR. These splice variants contain various structures in the extracellular domain, resulting in premature truncation (h alpha ENaCx), 19-amino acid deletion (h alpha ENaC-19), and 22-amino acid insertion (h alpha ENaC + 22). Wild-type h alpha ENaC and splice variants were functionally characterized in Xenopus oocytes by coexpression with hENaC beta- and gamma-subunits. Unlike wild-type h alpha ENaC, undetectable or substantially reduced amiloride-sensitive currents were observed in oocytes expressing these splice variants. Wild-type h alpha ENaC was the most abundantly expressed h alpha ENaC mRNA species in all tissues in which its expression was detected. These findings indicate that the extracellular domain is important to generate structural and functional diversity of h alpha ENaC and that alternative splicing may play a role in regulating hENaC activity.

Amino Acid Sequence↗

PubMed1997

Human tenascin-C: identification of a novel type III repeat in oral cancer and of novel splice variants in normal, malignant and reactive oral mucosae.

Tenascin-C is a mosaic, linear glycoprotein that is up-regulated during many normal and pathological processes involving either cell migration or tissue morphogenesis, such as invasion of malignant cells and wound healing. Human tenascin-C contains 8 consecutive type III fibronectin (TNCfn) domains that are involved in alternative splicing and potentially generate a large number of isoforms that code for tenascin-C proteins with subtly different functions. Human tenascin-C splice variants were investigated by RT-PCR in a range of normal and pathological oral mucosal tissues. A novel, 9th human TNCfn domain involved in alternative splicing was identified. It shares 70% nucleic acid and 55% protein sequence homology with chicken TNCfn-ad2. As in avians, this novel repeat was located between TNCfn-B and TNCfn-ad1 and accordingly was designated human TNCfn-ad2. Human TNCfn-ad2 was detected in only 2 of 10 oral cancers. However, TNCfn-ad2 was absent from 40 normal, reactive, pre-malignant and other oral mucosal specimens investigated. Previous studies have described 8 splice variant transcripts for human tenascin-C. By systematic investigation we identified further novel splice variants for human tenascin-C. Furthermore, our results indicate that many potential splice variants probably do not exist in the tissues investigated. Thus, we have demonstrated that human tenascin-C transcripts generate a complex but selected repertoire of different alternative splice products.

Alternative Splicing↗