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Survivin 2α: a novel Survivin splice variant expressed in human malignancies
Hugo Caldas, Laura E Honsey, Rachel A Altura
Molecular Cancer , 2005, DOI: 10.1186/1476-4598-4-11
Abstract: In the present study, we identify and characterize a novel survivin isoform that we designate survivin 2α. Structurally, the transcript consists of 2 exons: exon 1 and exon 2, as well as a 3' 197 bp region of intron 2. Acquisition of a new in-frame stop codon within intron 2 results in an open reading frame of 225 nucleotides, predicting a truncated 74 amino acid protein. Survivin 2α is expressed at high levels in several malignant cell lines and primary tumors. Functional assays show that survivin 2α attenuates the anti-apoptotic activity of survivin. Subcellular localization and immunoprecipitation of survivin 2α suggests a physical interaction with survivin.We characterized a novel survivin splice variant that we designated survivin 2α. We hypothesize that survivin 2α can alter the anti-apoptotic functions of survivin in malignant cells. Thus survivin 2α may be useful as a therapeutic tool in sensitizing chemoresistant tumor cells to chemotherapy.Alternative splicing is estimated to occur in 40–60% of all human genes, accounting for some of the discrepancies between the large number of known proteins and the three-fold lower number of human genes in the genome. Alternative splicing generates a multitude of isoforms that have overlapping but distinct functions during embryonic development and that also contribute to maintaining homeostasis in adult differentiated tissues (reviewed in [1]). Alternative splice forms of key proteins in cancer, TP53, MDM2 [2] and c-MYC [3], have been shown to play a role in oncogenesis.Survivin was originally identified by structural homology to IAPs in human B-cell lymphoma [4]. It is composed of a single BIR domain and an extended carboxy-terminal coiled coil domain [5]. Transcription from the Survivin locus gives rise to alternatively spliced transcripts identified in both human and mice [6-8]. To date, three alternatively spliced isoforms have been described in humans [6-8]. Survivin-2B is generated by the insertion of an alternat
Genomic organisation and alternative splicing of mouse and human thioredoxin reductase 1 genes
Simone A Osborne, Kathryn F Tonissen
BMC Genomics , 2001, DOI: 10.1186/1471-2164-2-10
Abstract: The human TXNRD1 gene spans 100 kb of genomic DNA organised into 16 exons and the mouse Txnrd1 gene has a similar exon/intron arrangement. We also analysed the alternative splicing patterns displayed by the mouse and human thioredoxin reductase 1 genes and mapped the different mRNA isoforms with respect to genomic organisation. These isoforms differ at the 5' end and encode putative proteins of different molecular mass. Genomic DNA sequences upstream of mouse exon 1 were compared to the human promoter to identify conserved elements.The human and mouse thioredoxin reductase 1 gene organisation is highly conserved and both genes exhibit alternative splicing at the 5' end. The mouse and human promoters share some conserved sequences.The thioredoxin system is comprised of thioredoxin (Trx) and thioredoxin reductase (TR) and plays an important role in maintaining the redox state of the cell [1]. This system is involved in many cellular functions including synthesis of deoxyribonucleotides [2], redox control of transcription factors [3], protection against oxidative stress [4], cell growth and cancer [5]. The reduced form of Trx is maintained by TR, an enzyme that contains a redox active disulphide group and a FAD molecule that uses the reducing power of NADPH [6,7]. Presently, there are three recognised forms of mammalian thioredoxin reductase: TR1, TR3 and TGR, that share a similar domain organisation and all contain selenocysteine [8-14]. TR1 was the first thioredoxin reductase to be characterised and is known as the cytosolic form [8]. TR3 is the mitochondrial form and is also known as TrxR2 [9-11]. TGR, previously known as the novel form of thioredoxin reductase (or TR2) functions as a Trx and GSSG reductase [11,12]. In this study, we focus upon the cytosolic form of mouse thioredoxin reductase (mTR1) and human thioredoxin reductase (hTR1). The mouse Txnrd1 gene is located on chromosome 10 and yields a cDNA sequence approximately 3200 bp long. This sequence encodes a
The Human Splice Variant Δ16HER2 Induces Rapid Tumor Onset in a Reporter Transgenic Mouse  [PDF]
Cristina Marchini,Federico Gabrielli,Manuela Iezzi,Santa Zenobi,Maura Montani,Lucia Pietrella,Cristina Kalogris,Anna Rossini,Valentina Ciravolo,Lorenzo Castagnoli,Elda Tagliabue,Serenella M. Pupa,Piero Musiani,Paolo Monaci,Sylvie Menard,Augusto Amici
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0018727
Abstract: Several transgenic mice models solidly support the hypothesis that HER2 (ERBB2) overexpression or mutation promotes tumorigenesis. Recently, a HER2 splice variant lacking exon-16 (Δ16HER2) has been detected in human breast carcinomas. This alternative protein, a normal byproduct of HER2, has an increased transforming potency compared to wild-type (wt) HER2 receptors. To examine the ability of Δ16HER2 to transform mammary epithelium in vivo and to monitor Δ16HER2-driven tumorigenesis in live mice, we generated and characterized a mouse line that transgenically expresses both human Δ16HER2 and firefly luciferase under the transcriptional control of the MMTV promoter. All the transgenic females developed multifocal mammary tumors with a rapid onset and an average latency of 15.11 weeks. Immunohistochemical analysis revealed the concurrent expression of luciferase and the human Δ16HER2 oncogene only in the mammary gland and in strict correlation with tumor development. Transgenic Δ16HER2 expressed on the tumor cell plasma membrane from spontaneous mammary adenocarcinomas formed constitutively active homodimers able to activate the oncogenic signal transduction pathway mediated through Src kinase. These new transgenic animals demonstrate the ability of the human Δ16HER2 isoform to transform “per se” mammary epithelium in vivo. The high tumor incidence as well as the short latency strongly suggests that the Δ16HER2 splice variant represents the transforming form of the HER2 oncoprotein.
Characteristics of CD44 alternative splice pattern in the course of human colorectal adenocarcinoma progression  [cached]
Bánky Balázs,Rásó-Barnett Lívia,Barbai Tamás,Tímár József
Molecular Cancer , 2012, DOI: 10.1186/1476-4598-11-83
Abstract: Background CD44 is considered as ‘a’ metastasis associated gene, despite the fact that it is an umbrella term for a group of molecules produced from a single gene by alternative splicing. However, little consideration is given to the above in the literature of colorectal carcinomas as well as other tumour types, leading to confusion and contradictory results about its possible role in tumour progression. Methods We compared the CD44 alternative splice pattern (ASP) of three genetically different human colorectal cancer cell lines (HT25, HT29, HCT116) using a series of PCR reactions and next- generation sequencing method, as well as identified a colorectal adenocarcinoma specific CD44 ASP. This ASP was further investigated in terms of its qualitative and quantitative stability in our experimental iso- and xenograft mouse models for colorectal cancer progression. A complex preclinical experimental set-up was established to separately test the different steps of tumour progression and the role of tumour microenvironment, respectively, focusing on the role of ‘CD44’ in this process. Results We managed to present a colorectal cancer-specific CD44 ASP, which remained unchanged from cell lines throughout primary tumour formation and metastatic progression. Furthermore, we report a unique roster of all expressed CD44 variant isoforms characteristic to colorectal cancer. Finally, on quantitative assessment of the variable exons v3 and v6, higher co-expression levels were found to be characteristic to metastatically potent tumour cells. Conclusion Particular CD44 variant isoforms seem to act as “metastasis genes” via tumour microenvironment-driven shifts in v3 and v6 expressions. However, this function may just affect a minority of tumour subclones. This fact and the huge potential number of different CD44 splice variants that can contain v3 and v6 domains can explain incoherence of clinical studies regarding functional asessment of CD44 variants, as well as diminish the chances of using CD44 variants for predictive purpose.
An Intron-Retaining Splice Variant of Human Cyclin A2, Expressed in Adult Differentiated Tissues, Induces a G1/S Cell Cycle Arrest In Vitro  [PDF]
Arata Honda, Yannick Valogne, Myriam Bou Nader, Christian Bréchot, Jamila Faivre
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0039249
Abstract: Background Human cyclin A2 is a key regulator of S phase progression and entry into mitosis. Alternative splice variants of the G1 and mitotic cyclins have been shown to interfere with full-length cyclin functions to modulate cell cycle progression and are therefore likely to play a role in differentiation or oncogenesis. The alternative splicing of human cyclin A2 has not yet been studied. Methodology/Principal Findings Sequence-specific primers were designed to amplify various exon–intron regions of cyclin A2 mRNA in cell lines and human tissues. Intron retaining PCR products were cloned and sequenced and then overexpressed in HeLa cells. The subcellular localization of the splice variants was studied using confocal and time-lapse microscopy, and their impact on the cell cycle by flow cytometry, immunoblotting and histone H1 kinase activity. We found a splice variant of cyclin A2 mRNA called A2V6 that partly retains Intron 6. The gene expression pattern of A2V6 mRNA in human tissues was noticeably different from that of wild-type cyclin A2 (A2WT) mRNA. It was lower in proliferating fetal tissues and stronger in some differentiated adult tissues, especially, heart. In transfected HeLa cells, A2V6 localized exclusively in the cytoplasm whereas A2WT accumulated in the nucleus. We show that A2V6 induced a clear G1/S cell cycle arrest associated with a p21 and p27 upregulation and an inhibition of retinoblastoma protein phosphorylation. Like A2WT, A2V6 bound CDK2, but the A2V6/CDK2 complex did not phosphorylate histone H1. Conclusion/Significance This study has revealed that some highly differentiated human tissues express an intron-retaining cyclin A2 mRNA that induced a G1/S block in vitro. Contrary to full-length cyclin A2, which regulates cell proliferation, the A2V6 splice variant might play a role in regulating nondividing cell states such as terminal differentiation or senescence.
Identification of a New Splice Variant of the Human ABCC6 Transporter
Maria Francesca Armentano,Angela Ostuni,Vittoria Infantino,Vito Iacobazzi,Maria Antonietta Castiglione Morelli,Faustino Bisaccia
Biochemistry Research International , 2008, DOI: 10.1155/2008/912478
Abstract: ABCC6 is a member of the adenosine triphosphate-binding cassette (ABC) gene subfamily C that encodes a protein (MRP6) involved in active transport of intracellular compounds to the extracellular environment. Mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), an autosomal recessive disorder of the connective tissue characterized by progressive calcification of elastic structures in the skin, the eyes, and the cardiovascular system. MRP6 is codified by 31 exons and contains 1503 amino acids. In addition to a full-length transcript of ABCC6, we have identified an alternatively spliced variant of ABCC6 from a cDNA of human liver that lacks exons 19 and 24. The novel isoform was named ABCC6 Δ19Δ24. PCR analysis from cDNA of cell cultures of primary human hepatocites and embryonic kidney confirms the presence of the ABCC6Δ19Δ24 isoform. Western blot analysis of the embryonic kidney cells shows a band corresponding to the molecular weight of the truncated protein.
Skipping of Exons by Premature Termination of Transcription and Alternative Splicing within Intron-5 of the Sheep SCF Gene: A Novel Splice Variant  [PDF]
Siva Arumugam Saravanaperumal, Dario Pediconi, Carlo Renieri, Antonietta La Terza
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0038657
Abstract: Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (?) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as ‘soluble’ isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a ‘novel’ mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D175G site which is necessary to produce ‘soluble’ form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6–9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (?) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals.
Expression of a splice variant of the platelet-activating factor receptor transcript 2 in various human cancer cell lines  [PDF]
Ibtissam Youlyouz,Emmanuelle Magnoux,Laurence Guglielmi,Yves Denizot
Mediators of Inflammation , 2002, DOI: 10.1080/09629350210000015755
Abstract: Platelet-activating factor receptor (PAF-R) transcripts were analysed by reverse transcriptase-polymerase chain reaction in five human cancer cell lines derived from the breast (BT20, SKBR3 and T47D cells), the pancreas (Miapaca cells) and the bladder (5637 cells) in order to confirm the existence of a splice variant of the PAF-R transcript 2. After cloning and sequencing, we confirmed its existence in all cell lines. It consisted of the PAF-R transcript 2 lengthening with 82 nucleotides from the 3' end of exon 1 of the PAF-R gene. The role of this elongated form of the tissue-type PAF-R transcript in cell physiology remains to be elucidated.
Identification of a truncated splice variant of IL-18 receptor alpha in the human and rat, with evidence of wider evolutionary conservation  [PDF]
Chris S. Booker,David R. Grattan
PeerJ , 2015, DOI: 10.7717/peerj.560
Abstract: Interleukin-18 (IL-18) is a pro-inflammatory cytokine which stimulates activation of the nuclear factor kappa beta (NF-κB) pathway via interaction with the IL-18 receptor. The receptor itself is formed from a dimer of two subunits, with the ligand-binding IL-18Rα subunit being encoded by the IL18R1 gene. A splice variant of murine IL18r1, which has been previously described, is formed by transcription of an unspliced intron (forming a ‘type II’ IL18r1 transcript) and is predicted to encode a receptor with a truncated intracellular domain lacking the capacity to generate downstream signalling. In order to examine the relevance of this finding to human IL-18 function, we assessed the presence of a homologous transcript by reverse transcription-polymerase chain reaction (RT-PCR) in the human and rat as another common laboratory animal. We present evidence for type II IL18R1 transcripts in both species. While the mouse and rat transcripts are predicted to encode a truncated receptor with a novel 5 amino acid C-terminal domain, the human sequence is predicted to encode a truncated protein with a novel 22 amino acid sequence bearing resemblance to the ‘Box 1’ motif of the Toll/interleukin-1 receptor (TIR) domain, in a similar fashion to the inhibitory interleukin-1 receptor 2. Given that transcripts from these three species are all formed by inclusion of homologous unspliced intronic regions, an analysis of homologous introns across a wider array of 33 species with available IL18R1 gene records was performed, which suggests similar transcripts may encode truncated type II IL-18Rα subunits in other species. This splice variant may represent a conserved evolutionary mechanism for regulating IL-18 activity.
A Novel Human TPIP Splice-Variant (TPIP-C2) mRNA, Expressed in Human and Mouse Tissues, Strongly Inhibits Cell Growth in HeLa Cells  [PDF]
Rasmi Rekha Mishra, Jitendra Kumar Chaudhary, Gagan Deep Bajaj, Pramod C. Rath
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0028433
Abstract: Alternative splicing of mRNAs is known to involve a major regulation of gene expression at RNA level in mammalian cells. The PTEN (Phosphatase and TENsin homologue deleted from the human chromosome 10), TPTE (Transmembrane Phosphatase with TEnsin homology) and TPIP (TPTE and PTEN homologous Inositol lipid Phosphatase) belong to a family of dual-specific lipid and protein phosphatases. PTEN is a well characterized tumor suppressor, which plays crucial role in cell survival, cell cycle regulation, cell proliferation as well as adhesion, motility and migration of cells. The C2-domain of PTEN is essential for PTEN-functions. We have isolated a novel 1019 bp human TPIP cDNA (TPIP-C2) from a human testis cDNA library. In silico analysis of the cDNA revealed that it is produced from the TPIP-locus on the human chromosome 13 by alternative RNA-splicing. It has a unique 5′-Alu sequence, a LINE sequence followed by a 582 bp Open Reading Frame (ORF) encoding a 193 aa polypeptide with a partial phosphatase domain and a C2-domain. TPIP-C2 mRNA is expressed in human testis and in mouse tissues. Mouse testis and brain showed higher levels of TPIP-C2 mRNA in comparison to the heart, liver and kidney under normal physiological conditions. TPIP-C2 mRNAs from human and mouse testes show extensive sequence identity. Over-expression of TPIP-C2 cDNA in HeLa cells strongly (up to 85%) inhibited cell growth/proliferation and caused apoptosis in a caspase 3-dependent manner. These findings suggest for the first time that a TPIP splice-variant mRNA with a partial phosphatase domain and a C2-domain is expressed in cells and tissues of human and murine origins under normal physiological conditions. Inhibition of cell growth/proliferation and induction of apoptosis by overexpression of TPIP-C2 mRNA in HeLa cells suggest that it may be involved in negative regulation of cell growth/proliferation.
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