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PubMed1996

Alternate promoters and alternate splicing of human tenascin-X, a gene with 5' and 3' ends buried in other genes.

Tenascin-X (TN-X) is an extracellular matrix protein encoded by a large gene that overlaps the steroid 21-hydroxylase (P450c21) gene in the HLA locus on chromosome 6p21.3. This may be the most complex locus in the human genome identified to date, containing 13 overlapping transcription units in 160 kb of DNA. Previous studies determined the sequence of 39 TN-X exons, encoding a 12 kb open reading frame, but the promoter(s) of the gene had not been located. We identify the principal TN-X promoter and a previously unknown 5' untranslated exon that lies more than 10 kb upstream from the previously known exons. This promoter, which is substantially different from the promoter for TN-C, initiates transcription in human fetal adrenal and muscle, but expression in human NCI-H295 adrenocortical carcinoma cells is initiated by two other promoters lying further upstream. One of these is the same as the promoter for a recently identified Creb-related protein (Creb-rp), but transcripts initiated form this promoter in human adrenal NCI-H295 tumor cells are spliced differently from Creb-rp, and are largely retained in the nuclei of these cells. By analogy with the other two members of the tenascin family, TN-C and TN-R, it has been predicted that TN-X should undergo alternate splicing in its fibronectin-like domains. RACE cloning and RNase protection experiments reveal no such alternate splicing. The TN-X gene appears to be unique in having both its 5' and 3' ends buried in other genes.

Adrenal Glands↗

PubMed1993

Drosophila gamma-aminobutyric acid receptor gene Rdl shows extensive alternative splicing.

The Drosophila gamma-aminobutyric acid (GABA) receptor subunit gene Rdl was isolated on the basis of a mutant phenotype showing high levels of insensitivity to picrotoxinin and cyclodiene insecticides. Following analysis of two dissimilar cDNAs isolated from the locus, we report that Rdl undergoes extensive alternative splicing at two locations in the putative extracellular domain. At each location a choice is made between exons of the same size: "a" or "b" (23 amino acids long with two substitutions) and "c" or "d" (46 residues long with 10 substitutions). The function of these alternative exons remains unclear; however, exon d contains a putative site for casein kinase II phosphorylation. All possible combinations of exons (a with c or d and b with c or d) were found in RNA isolated from early embryos. This is the first demonstration of alternative splicing in a GABA receptor gene from invertebrates.

Alternative Splicing↗

PubMed1999

Structure and alternative splicing of the gene encoding alpha1I, a human brain T calcium channel alpha1 subunit.

The structure of CACNA1I, the gene encoding alpha1I, a human brain T Ca2+ channel alpha1 subunit, was determined by comparison of polymerase chain reaction-amplified brain cDNA and genomic sequences. The gene consists of at least 36 exons spanning at least 115,168 basepairs of chromosome 22q12.3-13.2. The predicted protein has 2016 amino acids and 28 potential phosphorylation sites. Alternative splicing of the gene occurs at two sites: cassette exon 9 and an alternative acceptor in exon 33. Molecular diversity generated by alternative splicing and post-translational modification of this and other members of the T alpha1 subunit gene family may account for the observed heterogeneity of T currents in central neurons.

Alternative Splicing↗

PubMed2007

Differential signal transduction of alternatively spliced FGFR2 variants expressed in human mammary epithelial cells.

Gene amplification and protein overexpression of fibroblast growth factor receptor 2 (FGFR2) characterize the SUM-52 breast cancer cell line developed in our laboratory. SUM-52 cells express nine distinct alternatively spliced isoforms of FGFR2. Among these isoforms are two otherwise identical FGFR2 variants that express either the C1 or C3 carboxyl terminus. FGFR2-C3 variants are not normally expressed by human mammary epithelial (HME) cells, and we have shown that overexpression of FGFR2-C3 in HME cells results in potent transformation. In particular, FGFR2-C3 expression leads to robust levels of constitutively tyrosine phosphorylated FRS2 in the absence of ligand stimulation. In contrast, overexpressed FGFR2-C1 requires constant stimulation with exogenous keratinocyte growth factor (KGF) to mimic the signaling capability of FGFR2-C3. However, activation of FRS2 that results from KGF-stimulated FGFR2-C1 signaling is transient and is associated with a mobility shift of FRS2 not observed when this signaling molecule is activated by the C3 isoform of FGFR2. Mutation of the only tyrosine phosphorylated site present in the C1 terminus and absent from C3, Tyr769, did not yield a receptor that rivaled the potent signaling of FGFR2-C3. We therefore conclude that aberrant expression of alternatively spliced isoforms of FGFR2 with the C3 carboxyl terminus in the SUM-52 breast cancer cells results in sustained activation of signal transduction leading to transformation.

Alternative Splicing↗

PubMed2006

Molecular analyses of KCNQ1-5 potassium channel mRNAs in rat and guinea pig inner ears: expression, cloning, and alternative splicing.

CONCLUSION: Expression of neuronal Kcnq gene family transcripts in the inner ear provides further evidence for cochlear M-type currents and for complex molecular heterogeneities of voltage-gated potassium channels composed of various KCNQ subunits and/or alternative splice variants. Furthermore, important roles in regulation of cellular excitability in the auditory system, and hearing disorders related to (hyper)excitability, e.g. tinnitus, are implied. BACKGROUND: Voltage-gated potassium channels play key roles in hearing, as evidenced by deafness resulting from disruption of genes encoding, for example, KCNQ1 or KCNQ4 subunits. Other members of the Kcnq gene family (Kcnq2, 3, and 5) are the molecular correlates of M currents, which regulate neuronal excitability. The expression of the latter has not previously been thoroughly investigated in the inner ear. OBJECTIVE: The aim of this study was to identify genetic correlates of M currents, previously identified in cochlear hair cells by electrophysiological methods. MATERIALS AND METHODS: Expression of Kcnq genes was investigated by reverse transcription-polymerase chain reaction (RT-PCR) using subtype-specific primers with total RNA isolated from whole guinea pig or rat cochlea as template. PCR products were confirmed by direct DNA sequencing. RESULTS: All members of the Kcnq family were expressed in guinea pig and rat cochlea. Cochlear expression of Kcnq2 exhibited two alternatively spliced forms, lacking exons 8, 15a, and 8, 12a, 15a, respectively. Novel molecular sequence data, e.g. guinea pig Kcnq cDNA sequences, were deposited in GenBank (AY684985-AY684990).

Alternative Splicing↗

PubMed2002

Baboon placentas express soluble and membrane-bound Paan-AG proteins encoded by alternatively spliced transcripts of the class Ib major histocompatibility complex gene, Paan-AG.

The human class Ib major histocompatibility complex (MHC) molecule, HLA-G, is unique in its limited polymorphism, high expression in the placenta and generation of multiple transcripts by alternative splicing. The proteins encoded by these transcripts are believed to modulate maternal-fetal immunological relationships during pregnancy. The baboon placenta contains messages encoded by a novel MHC gene, Paan-AG, which is evolutionarily related to the HLA-A locus, but shares unique characteristics with HLA-G. In this study, we show that the Paan-AG message is alternatively spliced to generate at least seven transcripts. One of these transcripts retains intron 4 and encodes a soluble glycoprotein with three external domains and a unique 21-amino-acid sequence at the carboxyl terminus, similar to soluble HLA-G1. This glycoprotein was detected in first trimester placental villous cytotrophoblast and syncytiotrophoblast, and in extravillous cytotrophoblast cells in the basal plate in term placenta. Four of the transcripts ( Paan-AG1, Paan-AG2, Paan-AG3, Paan-AG4) encode membrane-bound class Ib MHC glycoprotein isoforms. Paan-AG1 protein expression was similar to that of sPaan-AG, while Paan-AG2 protein was not detected in these tissues. The other two transcripts ( Paan-AGx and Paan-AGxi) contain a truncated exon 3 and multiple stop codons. Paan-AG1 and Paan-AGx transcripts were detected in a number of non-placental tissues, but these transcripts contained multiple stop codons. Because of the structural similarities and common features of organ-specific expression and splicing of the message, studies on Paan-AG may be of value in dissecting the functions of the class Ib proteins in human pregnancy.

Alternative Splicing↗

PubMed1995

Alternative splicing of the neurofibromatosis 1 gene correlates with growth patterns and neuroendocrine properties of human small-cell lung-carcinoma cells.

Two distinct transcripts, type I and type II, of the neurofibromatosis I (NFI) gene are generated by alternative splicing in the region corresponding to the gene's GTPase-activating protein-related domain (GRD). Relative expression levels of these 2 transcripts were previously correlated to neural differentiation. Since small-cell lung carcinoma (SCLC) often exhibits neuroendocrine properties, we analyzed the type-I to type-II mRNA ratio in 15 SCLC cell lines, using reverse transcriptase and polymerase chain reaction methods. The type-I mRNA was predominant in 10 cell lines; 8 of them grew as floating aggregates in culture and had high L-dopa decarboxylase (DDC) activity. The other 5 lines predominantly expressed type-II mRNA, adhered to the culture substrate, and expressed low or undetectable levels of neural cell-adhesion molecule (NCAM) antigen and DDC activity. N2+, one of the subclones of NCI-N417 cells, exhibited a higher type-I to type-II ratio after the cells had adhered to a laminin-coated plate and had emitted neurite-like processes. These findings provide evidence that alternative splicing patterns of NFI mRNA correlate with the mechanisms that regulate the growth patterns and neuroendocrine properties of SCLC cells in vitro.

Alternative Splicing↗

PubMed1991

Alternative splicing and genomic structure of the Wilms tumor gene WT1.

The chromosome 11p13 Wilms tumor susceptibility gene WT1 appears to play a crucial role in regulating the proliferation and differentiation of nephroblasts and gonadal tissue. The WT1 gene consists of 10 exons, encoding a complex pattern of mRNA species: four distinct transcripts are expressed, reflecting the presence or absence of two alternative splices. Splice I consists of a separate exon, encoding 17 amino acids, which is inserted between the proline-rich amino terminus and the zinc finger domains. Splice II arises from the use of an alternative 5' splice junction and results in the insertion of 3 amino acids between zinc fingers 3 and 4. RNase protection analysis demonstrates that the most prevalent splice variant in both human and mouse is that which contains both alternative splices, whereas the least common is the transcript missing both splices. The relative distribution of splice variants is highly conserved between normal fetal kidney tissue and Wilms tumors that have intact WT1 transcripts. The ratio of these different WT1 mRNA species is also maintained as a function of development in the mouse kidney and in various mouse tissues expressing WT1. The conservation in structure and relative levels of each of the four WT1 mRNA species suggests that each encoded polypeptide makes a significant contribution to normal gene function. The control of cellular proliferation and differentiation exerted by the WT1 gene products may involve interactions between four polypeptides with distinct targets and functions.

Amino Acid Sequence↗

PubMed2001

Differentiation strategies of alternative splicing variants mRNA estrogen receptor-alpha.

From the clinical point of view recognizing the concentrations and type profile of isoforms could be of significant practical importance. The aim of the study is designed QRT-PCR reaction to assess profile of mRNA ER-alpha and their isoforms. Theoretical part of the study was made according to computer program and available Genbank database. To detect a isoform one of the primer was designed to hybridize within exon-border linked in alternative splicing. The study presents the differentiation strategies in isoforms coming about as the alternative splicing result. Designed oligonucleotide probes and primers allow to distinguish mRNA isoforms of ER-alpha and their quantification in assessed tissues.

Alternative Splicing↗

PubMed2005

Inactivation of the invasion inhibitory gene IIp45 by alternative splicing in gliomas.

The invasion inhibitory protein 45 (IIp45) we recently identified was underexpressed in glioblastoma multiforme, the most malignant form of glioma. The IIp45 gene is located at chromosome 1p36 where frequent deletions have been reported in various types of tumors, including gliomas, raising the possibility that IIp45 may be a classic tumor suppressor gene that can be inactivated by frequent point mutations. To test this hypothesis, we sequenced the IIp45 gene in 59 diffuse glioma samples of different grades and histologic subtypes and identified a possible point mutation or a rare polymorphism in only one sample (1.7%), suggesting that IIp45 is not a classic tumor suppressor gene such as p53. Instead, reverse transcription-PCR and subsequent sequencing results revealed a tumor-specific IIp45 spliced isoform (IIp45S) in 20 of 59 (34%) gliomas examined, particularly in glioblastoma multiformes, including native tissue samples (15 of 25; 60%) and cell lines (5 of 5; 100%). The alternative splicing event is independent of 1p36 deletion, which is not common in glioblastoma multiforme. The IIp45S transcript was not detected in any of 18 normal organs, including fetal and adult brain. We determined that the IIp45S isoform results from exclusion of IIp45 exon 7 and encodes a variant protein that carries a COOH terminus different from that of IIp45 due to a frame-shift mutation. IIp45S protein was undetectable in glioma tissues, although IIp45S mRNA was prevalent. We found that IIp45S, once translated, is rapidly degraded by an ubiquitin-proteasome mechanism. Thus, the IIp45 gene is inactivated by a tumor-specific alternative splicing that generates an aberrant and unstable IIp45 isoform in infiltrative gliomas.

Alternative Splicing↗

PubMed1999

The Drosophila melanogaster gene for the NADH:ubiquinone oxidoreductase acyl carrier protein: developmental expression analysis and evidence for alternatively spliced forms.

We have isolated the Drosophila melanogaster gene encoding the mitochondrial acyl carrier protein (mtACP), a subunit of NADH:ubiquinone oxidoreductase involved in de novo fatty acid synthesis in the mitochondrion. This gene expresses two distinct mature transcripts by alternative splicing, which encode mature polypeptides of 86 (mtACP1A) and 88 (mtACP1B) amino acids, respectively. Drosophila mtACP1 is 72% identical to mammalian mtACP, 47% identical to Arabidopsis thaliana mtACP, and 46% identical to Neurospora crassa mtACP. The most highly conserved region encompasses the site that binds pantetheine-4'-phosphate in all known ACPs. Southern analysis of genomic DNA and in situ hybridization to salivary gland chromosomes indicate that a single gene (mtacp1), located at 61F6-8, encodes the two isoforms of D. melanogaster mtACP1. Sequence analysis revealed that the gene contains four exons and that exons IIIA and IIIB are alternatively spliced. A P-element-induced loss-of-function mutation in the mtacp1 gene causes lethality, indicating that the gene is essential for viability. Developmental Northern analysis shows that mtacp1 is expressed at higher levels during late embryogenesis, in the pupa and in the adult. RNA in situ hybridization on embryos indicates that the mtacp1 gene is highly expressed in the tracheal system. Zygotic mtacp1 function is required for both male and female gametogenesis.

Acyl Carrier Protein↗

PubMed2001

The long form of CDK2 arises via alternative splicing and forms an active protein kinase with cyclins A and E.

We have reinvestigated the long form of cyclin-dependent kinase (CDK)2 that is expressed in many rodent cells. We show that the mRNA encoding CDK2L arises by alternative splicing and that the encoded protein can bind to, and be activated by, cyclins A and E. The complex of CDK2L with cyclin A has about half the specific activity of the equivalent CDK2-cyclin A complex. Also, CDK2L--cyclin A is inhibited to the same extent and by the same concentrations of p21(CIP1) as CDK2--cyclin A. The nucleotide sequences of intron V in the human and murine CDK2 genes, where the sequences encoding the 48-residue insert in CDK2L are located, show very high conservation in the position of the alternatively spliced exon and its surroundings. Despite this, we were not able to detect significant expression of CDK2L in human cell lines, although a low level is expressed in COS-1 cells from monkeys.

Alternative Splicing↗

PubMed2005

Ankyrin-G in skeletal muscle: tissue-specific alternative splicing contributes to the complexity of the sarcolemmal cytoskeleton.

Ankyrins are versatile adaptor proteins that join the spectrin-based cytoskeleton to transmembrane proteins, and have roles in organizing the microstructure of cell membranes. Molecular diversity of ankyrins in mammals arises from extensive alternative splicing of the products of three genes. There has been no systematic analysis of the diversity of expression of ankyrins-G, the widely expressed Ank3 gene products, in a complex tissue. We previously described Ank(G107), the first muscle-specific ankyrin-G. Here, we combined cDNA and database analyses to gain novel insight into the ankyrins-G of skeletal muscle. We find: (i) that Ank3 is composed of at least 53 exons, many of which are subject to tissue-specific splicing; (ii) five novel full-length cDNAs encoding two canonical (Ank(G197), Ank(G217)) and three small isoforms (Ank(G109), Ank(G128), Ank(G130)) bring to six the number of ankyrins-G expressed in skeletal muscle; (iii) a 76-residue insert in the C-terminal domain is a 'signature' for muscle ankyrins; (iv) variably spliced sequences of 17/18 and 195 residues increase diversity in the C-terminal domains. Comparison of endogenous ankyrins-G with in vitro translated cDNAs revealed that small ankyrins account for the majority of the immunoreactivity for ankyrin-G in soleus muscle. The small ankyrins, when expressed in vivo in the rat muscle, are all targeted to sarcolemmal costameres. Our results demonstrate the tissue-dependent alternative splicing of Ank3 in skeletal muscle and point to novel functions of small ankyrins-G in organizing microdomains of the plasma membrane.

Alternative Splicing↗

PubMed2005

Dynamic balance between activation and repression regulates pre-mRNA alternative splicing during heart development.

Cardiac troponin T (cTNT) exon 5 splicing is developmentally regulated such that it is included in embryonic but not adult heart. CUG-BP and ETR-3-like factor (CELF) proteins promote exon inclusion, whereas polypyrimidine tract binding protein (PTB) and muscleblind-like (MBNL) proteins repress inclusion. In this study, we addressed what happens to these regulatory proteins during heart development to shift the regulatory balance of cTNT alternative splicing. Using dominant-negative proteins, we found that both CELF and PTB activities are required for appropriate splicing in cardiomyocytes. Two CELF proteins, CUG-BP and ETR-3, are nuclear and cytoplasmic in embryonic heart but are down-regulated in adult heart concomitant with loss of exon inclusion. In contrast, PTB and MBNL1 are expressed throughout heart development. The patterns of cTNT splicing and expression of its regulatory factors are conserved between mouse and chicken. Thus, alternative splicing is determined by a balance between positive and negative regulation, and modulation of expression levels of auxiliary splicing regulators may drive developmental splicing changes. ETR-3 and CUG-BP proteins are also down-regulated in other tissues during development, suggesting that CELF proteins play a broad role in developmental splicing regulation.

Aging↗

PubMed2002

Regulation of alternative splicing by SRrp86 through coactivation and repression of specific SR proteins.

SRrp86 is an 86-kDa member of the SR protein superfamily that is unique in that it can alter splice site selection by regulating the activity of other SR proteins. To study the function of SRrp86, inducible cell lines were created in which the concentration of SRrp86 could be varied and its effects on alternative splicing determined. Here, we show that SRrp86 can activate SRp20 and repress SC35 in a dose-dependent manner both in vitro and in vivo. These effects are apparently mediated through direct protein-protein interaction, as pull-down assays showed that SRrp86 interacts with both SRp20 and SC35. Consistent with the hypothesis that relatively modest changes in the concentration or activity of one or more splicing factors can combinatorially regulate overall splicing, protein expression patterns of SRrp86, SRp20, and SC35 reveal that each tissue maintains a unique ratio of these factors. Regulation of SR protein activity, coupled with regulated protein expression, suggest that SRrp86 may play a crucial role in determining tissue specific patterns of alternative splicing.

3' Untranslated Regions↗

PubMed1999

Novel alternatively spliced isoforms of the neurofibromatosis type 2 tumor suppressor are targeted to the nucleus and cytoplasmic granules.

We cloned novel splice variants Mer150, Mer151 and Mer162 of the neurofibromatosis 2 (NF2) tumor suppressor, which demonstrate a tissue-specific and development-specific expression pattern. Isoform Mer150 is created by cryptic splicing from exon 8 to 14 and represents an N-terminal truncation of 259 residues. Mer151 is characterized by in-frame splicing out of several exons and a modified C-terminus due to a frameshift in exons 13+14 and premature termination. Mer162 represents a head-to-tail isoform resulting from in-frame skipping of exons 5-16. As a common feature, the alpha-helical domain and a variable proportion of the ERM homology domain are spliced out in these isoforms. To investigate differences in subcellular localization, we expressed epitope-tagged cDNA constructs of the wild-type NF2 as well as of the three alternatively spliced transcripts in NIH 3T3 cells by nuclear microinjection or lipid-mediated transfection. Subcellular localization of Mer151 in filopodia and ruffling membranes was similar to the wild-type NF2. Mer151, however, was targeted to the nucleus, which was not observed for wild-type NF2, Mer150 or Mer162. A putative nuclear localization signal created by alternative splicing was identified in Mer151. In contrast to Mer151, Mer150 and Mer162 were not found in regions of the plasma membrane, but localized to a granular intracellular compartment. The results suggest that the recently described actin-binding domain in exon 10, but not the presence or absence of exons 2+3, is relevant for subcellular targeting. Although the NF2 protein is known as a cytoskeletal linker, additional functions in a cytoplasmic compartment and in the nucleus may exist.

3T3 Cells↗

PubMed1990

Human ceruloplasmin. Tissue-specific expression of transcripts produced by alternative splicing.

Ceruloplasmin (CP) is a plasma glycoprotein that transports copper throughout the body. In previous studies (Yang, F., Naylor, S., Lum, J., Cutshaw, S., McCombs, J., Naberhaus, K., McGill, J., Adrian, G., Moore, C., Barnett, D., and Bowman, B. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 3277-3261), two CP cDNA clones, CP-1 and CP-2, from a human cDNA library, differed from each other by the presence or absence, respectively, of 12 nucleotide bases encoding a deduced sequence of Gly-Glu-Tyr-Pro near the carboxyl-terminal region of the ceruloplasmin molecule. Examination of genomic DNA demonstrates that the two CP mRNAs are produced from a single gene by alternative spliced patterns. The additional amino acids deduced in CP-1 are products of alternative splicing within an intron of the CP gene at a site 12 nucleotide bases 3' to the commonly used site of CP-2. The CP-1 mRNA transcript encoding four extra amino acids appeared as a minor species accompanying CP-2 mRNA in placenta and chondrocytes. CP-1 mRNA was the predominant CP transcript in a lymphoblastic leukemia cell line, CEM. The mRNA examined from other tissues contained only CP-2 mRNA transcripts. These findings predict that alternative RNA splicing may lead to the differential expression of CP genomic sequences and produce alternate isoforms from a single CP gene in specific tissues.

Amino Acid Sequence↗

PubMed1999

Alternative splicing creates sex-specific transcripts and truncated forms of the furin protease in the parasite Dirofilaria immitis.

Many extracellular proteins are activated by specific cleavage with an endoprotease. In nematodes, several proteins are cleaved after RX(K/R)R, the recognition site for the subtilisin-like proprotein convertases, furin and blisterase. To characterize furin in the parasitic nematode Dirofilaria immitis, we determined the sequence of the difur gene and its multiple transcripts. The gene spans 11 kb; encodes 16 exons and has a complex pattern of alternative splicing which generates at least 16 distinct mRNAs. The major transcript is a 4.4 kb mRNA which codes for a protein of 834 aa with an unusually long prodomain of 254 aa. Sex-specific splice variants of difur were observed by RT-PCR. The three female-specific and five male-specific transcripts are the first reported examples of sex-specific splicing in parasitic nematodes. This suggests that nematodes have sex-specific factors which regulate RNA splicing. Other splice variants are predicted to alter the phosphorylation and localization of the protease. Alternative splicing after the prodomain encodes a truncated protein that may be an inhibitor and/or substrate of Difurin.

Alternative Splicing↗