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Results for “Alternative Splicing”

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PubMed1992

Identification and characterization of alternatively spliced fibronectin mRNAs expressed in early Xenopus embryos.

Sequence analysis of cDNA clones encoding fibronectin (FN) from Xenopus laevis reveals extensive amino acid identities with other vertebrate FNs, including the presence of the Arg-Gly-Asp (RGD) cell attachment site in type III-10 and of a second, cell-binding site (EILDV) in the alternative spliced V region of the protein. These cDNAs have been used to study the expression of FN mRNAs during early development. Overall, levels of maternal FN mRNA remain constant until the mid- to late-gastrula stage when the accumulation of new FN transcripts is first apparent. RNase protection analyses reveal that the pattern of FN alternative splicing is similar to that reported for other species and does not change with the shift from maternal to zygotic mRNA expression. The cellular forms of the FN protein predominate in the early embryo with the EIIIA and EIIIB exons included in most mRNAs at this time. A comparison of V-region alternative splicing between embryonic and adult liver RNAs indicates a segment of 345 nucleotides that can be either completely excluded or included in mature FN transcripts but there is no evidence for additional V-region variants. Maternal mRNAs encoding alternatively spliced forms of FN can be specifically eliminated from Xenopus oocytes following the injection of antisense oligodeoxynucleotides into the cytoplasm, thereby making it possible to analyze the structure, composition, and function of FN mRNAs in early embryos.

Amino Acid Sequence↗

PubMed2005

ASF/SF2-regulated CaMKIIdelta alternative splicing temporally reprograms excitation-contraction coupling in cardiac muscle.

The transition from juvenile to adult life is accompanied by programmed remodeling in many tissues and organs, which is key for organisms to adapt to the demand of the environment. Here we report a novel regulated alternative splicing program that is crucial for postnatnal heart remodeling in the mouse. We identify the essential splicing factor ASF/SF2 as a key component of the program, regulating a restricted set of tissue-specific alternative splicing events during heart remodeling. Cardiomyocytes deficient in ASF/SF2 display an unexpected hypercontraction phenotype due to a defect in postnatal splicing switch of the Ca(2+)/calmodulin-dependent kinase IIdelta (CaMKIIdelta) transcript. This failure results in mistargeting of the kinase to sarcolemmal membranes, causing severe excitation-contraction coupling defects. Our results validate ASF/SF2 as a fundamental splicing regulator in the reprogramming pathway and reveal the central contribution of ASF/SF2-regulated CaMKIIdelta alternative splicing to functional remodeling in developing heart.

Alternative Splicing↗

PubMed1994

Endogenous p53 protein generated from wild-type alternatively spliced p53 RNA in mouse epidermal cells.

We previously demonstrated that a wild-type alternatively spliced p53 (p53as) RNA exists in mouse cultured cells and normal mouse tissues at approximately 25 to 33% of the level of the major p53 RNA form. The alternative RNA transcript is 96 nucleotides longer than the major transcript as a result of alternative splicing of intron 10 sequences. The protein expected to be generated from the p53as transcript is 9 amino acids shorter than the major p53 protein and has 17 different amino acids at the carboxyl terminus. We report here that p53as protein exists in nontransformed and malignant epidermal cells and is localized to the nucleus. In addition, p53as protein is preferentially expressed during the G2 phase of the cell cycle and in cells with greater than G2 DNA content compared with the major p53 protein, which is preferentially expressed in G1. The p53as immunoreactivity is elevated and shifted to the G1 phase of the cell cycle following actinomycin D treatment of nontransformed cells but not malignant cells. In view of the dimerization and tetramerization of p53 protein which may be necessary for its DNA binding and transcriptional activation activities, the presence of p53as protein in cells has important implications for understanding the physiological function(s) of the p53 gene.

Alternative Splicing↗

PubMed2001

Alternate splicing of the shal gene and the origin of I(A) diversity among neurons in a dynamic motor network.

The pyloric motor system, in the crustacean stomatogastric ganglion, produces a continuously adaptive behavior. Each cell type in the neural circuit possesses a distinct yet dynamic electrical phenotype that is essential for normal network function. We previously demonstrated that the transient potassium current (I(A)) in the different component neurons is unique and modulatable, despite the fact that the shal gene encodes the alpha-subunits that mediate I(A) in every cell. We now examine the hypothesis that alternate splicing of shal is responsible for pyloric I(A) diversity. We found that alternate splicing generates at least 14 isoforms. Nine of the isoforms were expressed in Xenopus oocytes and each produced a transient potassium current with highly variable properties. While the voltage dependence and inactivation kinetics of I(A) vary significantly between pyloric cell types, there are few significant differences between different shal isoforms expressed in oocytes. Pyloric I(A) diversity cannot be reproduced in oocytes by any combination of shal splice variants. While the function of alternate splicing of shal is not yet understood, our studies show that it does not by itself explain the biophysical diversity of I(A) seen in pyloric neurons.

Alternative Splicing↗

PubMed1999

Expression of alternatively spliced mdm2 transcripts correlates with stabilized wild-type p53 protein in human glioblastoma cells.

A puzzling finding in various human tumors, including glioblastoma multiforme (GBM), is the stabilization of wild-type (wt) p53 protein. The biological significance of this phenomenon and the mechanism by which it occurs are unexplained. Recent reports have revealed that mdm2 exerts its negative regulation on the p53 signal by directly binding p53 protein and thereby instigating its proteasomal degradation. mdm2 has been shown to exist in alternatively spliced forms in human ovarian and bladder carcinomas, and recently in GBM, with loss or disruption of its p53 binding domain. Here we report that alternatively spliced transcripts of mdm2 are present in 7 of 16 human GBM primary cell cultures and in the established GBM cell lines LN 229 and LN 18. Sequencing demonstrated loss of the amino terminal p53 binding domain in these alternatively spliced mdm2 transcripts, and an out-of-frame splicing in the majority of cases. A significant correlation between the presence of mdm2 splice variants and increased expression of wt p53 protein was observed. Furthermore, in the presence of an mdm2 splice variant, wt p53 stabilization occurred despite coincident MDM2 amplification. Our findings suggest that wt p53 protein stabilization may arise as a consequence of alternative splicing of mdm2. Such a mechanism might account for wt p53 protein accumulation in GBM cells, even in the presence of MDM2 gene amplification.

Binding Sites↗

PubMed1991

The chicken tropomyosin 1 gene generates nine mRNAs by alternative splicing.

Skeletal muscle beta-tropomyosin, smooth muscle alpha-tropomyosin, and a low molecular weight fibroblast tropomyosin are generated by alternatively splicing RNA transcripts of the chicken tropomyosin 1 (TM 1) gene (Forry-Schaudies, S., Maihle, N. J., and Hughes, S. H. (1990) J. Mol. Biol. 211; 321-330). Two novel tropomyosin cDNAs that derive from mRNAs of the TM 1 gene have been isolated from a chicken embryo brain cDNA library. Brain cDNA BRT-1 is 2.2 kilobases in length and encodes 283 amino acids. It is identical to skeletal muscle beta-tropomyosin from amino acids 1 to 258. The sequence 3' of this point is unique to BRT-1; a comparison to genomic sequence indicates that a new carboxyl-terminal exon is used to generate this sequence. 1.4-kilobase brain cDNA BRT-2 contains sequences found in both fibroblast cDNA FT-beta (5'-end) and skeletal muscle cDNA SKT-beta (3'-end). RNase and S1 nuclease assays using RNA samples from leg muscle, gizzard, fibroblasts, and brain indicate that the TM 1 gene expresses four additional tropomyosin RNAs by alternately splicing previously characterized exons. These results demonstrate that the chicken TM 1 gene encodes nine tropomyosin RNAs through the use of two promoters, two internal exons that are mutually exclusive, and three 3'-exons. Implications for the regulation of alternative splicing are discussed.

Amino Acid Sequence↗

PubMed1989

Cell type specific trans-acting factors are involved in alternative splicing of human fibronectin pre-mRNA.

ED-A and ED-B are facultative type III homologies of fibronectin, encoded by alternatively spliced exons, described in man and in rat. A hybrid alpha-globin-fibronectin minigene containing the ED-B region from the human gene has been transfected in human cell lines derived from various tissues, in order to study the processing of the generated precursor RNA in the different cell environments. In most tested lines the pre-RNA is alternatively spliced and produces two mature RNAs, with and without the ED-B exon, in different ratios that closely resemble the corresponding endogenous fibronectin RNAs. In a hepatoma cell line, Hep 3B, only one RNA is produced, in which the ED-B exon is absent; the same pattern of splicing is observed in liver. The data show that all the information required to produce accurate and regulated alternative splicing of the ED-B exon is contained in the fragment used and cell specific factors are necessary for the pre-RNA to be differentially spliced in the various cell lines. In contrast, expression in Hep 3B of a similar gene containing the ED-A area failed to reproduce the liver specific splicing pattern. Therefore regulation of ED-A processing is likely to involve different mechanisms to those responsible for control of ED-B splicing.

Base Sequence↗

PubMed1994

Targeted amplification of alternatively spliced transcripts of major histocompatibility complex class I heavy chain.

The RPMI1788 cell line was found to produce soluble form of HLA class I molecules (sHLA) constitutively, due at least in part to an alternative splicing mechanism in which exon 5 of HLA class I heavy chain transcripts is deleted. Reverse transcription-polymerase chain reaction (RT-PCR) of cytoplasmic RNA of RPMI1788 cells using a pair of primers (A,B) complementary to the conserved sequences of HLA class I exon 4 and 6 yielded almost exclusively the full-length class I heavy chain cDNA. In order to amplify the alternatively spliced transcripts, primer C corresponding to the 5' boundary conserved region of exon 6 juxtaposed with three conserved nucleotides in 3' boundary region of exon 4 was synthesized. Using the primers A and C the spliced transcripts of RPMI1788 cells can be selectively or preferentially amplified by RT-PCR with three different DNA polymerases. Cloning and sequencing of the resulting cDNA confirmed that the spliced transcript lacks exon 5. The targeted amplification method may be useful and important for studies with respect to the regulation of class I sHLA expression and the mechanism by which alternative splicing of HLA class I heavy chain mRNA is induced.

Alternative Splicing↗

PubMed1993

Enhanced activation of the human histone H2B promoter by an Oct-1 variant generated by alternative splicing.

POU homeodomain proteins are important regulators of ubiquitous as well as tissue-specific transcription. These factors include the broadly expressed octamer motif-binding protein Oct-1 and the related cell-specifically expressed protein Oct-2. These two proteins differ in the types of octamer motif-containing promoters they preferentially activate; Oct-1 can activate RNA polymerase II transcription from a small nuclear RNA promoter better than Oct-2, which can better activate an mRNA-type promoter. We describe a variant Oct-1-encoding cDNA resulting from two separate alternate splices of the human oct-1 primary transcript; these alternate splices were present in all cell lines tested. This cDNA encodes an amino-terminally and carboxyl-terminally truncated form of Oct-1, called Oct-1B, which retains the DNA-binding POU domain and acquires a unique 12-amino acid carboxyl-terminal extension. In a transient expression assay, Oct-1B displayed an enhanced ability compared to the larger form of Oct-1 (called Oct-1A in this report) to activate the human histone H2B promoter, an mRNA-type promoter where a natural octamer motif is involved in cell cycle dependent transcription. Thus, the ability of Oct-1 related proteins to activate a natural regulatory target can be influenced by alternative splicing.

Alternative Splicing↗

PubMed1997

Primary structure and developmental acidic to basic transition of 13 alternatively spliced mouse fast skeletal muscle troponin T isoforms.

Large samples of original cDNAs encoding neonatal and adult mouse fast skeletal muscle troponin T (fTnT) have been isolated and characterized. The results demonstrate expression relationships of 8 alternatively spliced exons of the fTnT gene and reveal the primary structure of as many as 13 fTnT isoforms that diverge into acidic and basic classes due to differential mRNA splicing in the N-terminal variable region. In the C-terminal variable region encoded by the mutually exclusive exons 16 and 17, the splicing pathway and structure of exon 16 appears to be adult fTnT-specific, suggesting an adaptation to the functional demands of mature fast skeletal muscle. The cloned cDNAs were expressed in E. coli as standards to identify a high M(r) to low M(r), acidic to basic fTnT isoform transition in postnatal developing skeletal muscles. Different from the developmental cardiac TnT switch generated by alternative splicing of a single exon, the fTnT isoform transition is an additive effect of alternative splicing of multiple N-terminal-coding exons, especially exons 4, 8 and fetal that are expressed at higher frequencies in the neonatal than in the adult muscle. The developmental fTnT isoform primary structure transition in both N- and C-terminal variable regions suggest a physiological importance of the apparently complex TnT isoform expression.

Acids↗

PubMed2006

RNA polymerase II C-terminal domain mediates regulation of alternative splicing by SRp20.

Previous studies have linked the C-terminal domain (CTD) of RNA polymerase II (pol II) with cotranscriptional precursor messenger RNA processing, but little is known about the CTD's function in regulating alternative splicing. We have examined this function using alpha-amanitin-resistant pol II CTD mutants and fibronectin reporter minigenes. We found that the CTD is required for the inhibitory action of the serine/arginine-rich (SR) protein SRp20 on the inclusion of a fibronectin cassette exon in the mature mRNA. CTD phosphorylation controls transcription elongation, which is a major contributor to alternative splicing regulation. However, the effect of SRp20 is still observed when transcription elongation is reduced. These results suggest that the CTD promotes exon skipping by recruiting SRp20 and that this contributes independently of elongation to the transcriptional control of alternative splicing.

Alternative Splicing↗

PubMed1997

Frequent association of alternative splicing of NER, a nuclear hormone receptor gene in cancer tissues.

We have detected frequent alternative splicing of a gene that encodes NER, a protein homologous to the retinoic acid receptors, in cancer cells. Western and immunohistochemical analyses disclosed accumulation of a large amount of the aberrant NER product, generated by alternative splicing that caused skipping of an exon corresponding to the DNA-binding domain, in the nucleoli of cells of cancer cell lines and primary cancer tissues. The aberrant protein was detected in 116 of 228 primary cancers developed in various tissues including breast and colon, but was absent in the corresponding normal tissues; it was also detected in 31 of 39 cancer cell lines. This observation may imply that the aberrant NER product has some relation to the development and/or progression of cancers in a variety of human tissues.

Alternative Splicing↗

PubMed1995

Alternative splicing of T cell receptor (TCR) alpha chain transcripts containing V alpha 1 or V alpha 14 elements.

Human acute lymphoblastic leukemia cell lines represent valuable tools to investigate distinct steps of the complex regulatory pathways underlying T cell receptor recombination and expression. A case in point are V delta 2D delta 3 and subsequent V delta 2D delta 3J alpha rearrangements observed in human leukemic pre-B cells as well as in normal lymphopoiesis. The functional expression of these unusual (VD) delta (JC) alpha hybrids is almost exclusively prevented by alternative splicing events. In this report we show that alternative splicing at cryptic splice donor sites within V elements is not a unique feature of hybrid TCR delta/alpha transcripts. Among seven V alpha families analyzed by RT-PCR, alternatively spliced products were observed in TCR alpha recombinations containing V alpha 1 or V alpha 14 elements. In contrast to normal peripheral blood cells and thymocytes, the leukemia cell line JM expressing functional V alpha 1J alpha 3C alpha transcripts lacked evidence of aberrant TCR alpha RNA species.

Alternative Splicing↗

PubMed2001

A CaMK IV responsive RNA element mediates depolarization-induced alternative splicing of ion channels.

Calcium regulation of gene expression is critical for the long-lasting activity-dependent changes in cellular electrical properties that underlie important physiological functions such as learning and memory. Cellular electrical properties are diversified through the extensive alternative splicing of ion channel pre-messenger RNAs; however, the regulation of splicing by cell signalling pathways has not been well explored. Here we show that depolarization of GH3 pituitary cells represses splicing of the STREX exon in BK potassium channel transcripts through the action of Ca2+/calmodulin-dependent protein kinases (CaMKs). Overexpressing constitutively active CaMK IV, but not CaMK I or II, specifically decreases STREX inclusion in the mRNA. This decrease is prevented by mutations in particular RNA repressor sequences. Transferring 54 nucleotides from the 3' splice site upstream of STREX to a heterologous gene is sufficient to confer CaMK IV repression on an otherwise constitutive exon. These experiments define a CaMK IV-responsive RNA element (CaRRE), which mediates the alternative splicing of ion channel pre-mRNAs. The CaRRE presents a unique molecular target for inducing long-term adaptive changes in cellular electrical properties. It also provides a model system for dissecting the effect of signal transduction pathways on alternative splicing.

Alternative Splicing↗

PubMed1998

High-level production of alternatively spliced soluble interleukin-6 receptor in serum of patients with adult T-cell leukaemia/HTLV-I-associated myelopathy.

We have previously shown, using human T-cell lymphocytotrophic virus-I (HTLV-I)-infected cell lines, that soluble interleukin-6 receptor (sIL-6R) is generated through an alternative splicing mechanism. In this study, we examined human sera for the presence of alternatively spliced soluble IL-6R (AS-sIL-6R). We produced a monoclonal antibody (mAb) recognizing the unique sequence of AS-sIL-6R peptide, generated by an altered reading frame. We also made recombinant AS-sIL-6R protein in Spodoptera frugiperda-9 (Sf-9) cells carrying baculovirus, which encoded altered sIL-6R or conventional IL-6R cDNA. mAbs specifically recognized AS-sIL-6R, but not conventional IL-6R, as demonstrated by Western blot analyses, fluorescence-activated cell sorter, immunofluorescence analyses and enzyme-linked immunosorbent assay (ELISA). We adapted an ELISA system and used it for detection of altered sIL-6R in sera from 23 healthy persons, 12 patients with adult T-cell leukaemia (ATL) and 13 patients with HTLV-I-associated myelopathy (HAM). Serum levels of AS-sIL-6R were 6.4 or 6.1 times greater in ATL (28.7+/-20.4 ng/ml, P<0.0001) and in HAM patients (27.5+/-12.1 ng/ml, P<0.0001) than in healthy individuals (4.5+/-2.1 ng/ml). High levels of AS-sIL-6R were also observed in plasma from rheumatoid arthritis patients and in persons with elevated levels of alanine aminotransferase (ALT), antinuclear antibody (ANA), or alpha-fetoprotein (AFP). However, in human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV) or hepatitis C virus (HCV)-infected individuals, AS-sIL-6R levels were not elevated. In this study, we confirmed that AS-sIL-6R is indeed present in human sera. These observations suggest that alternative splicing of IL-6R mRNA is of consequence in ATL, HAM and in some autoimmune diseases. The HTLV-I-infected T cells appeared to play an important role in AS-sIL-6R production.

Adult↗

PubMed1995

Variant Max protein, derived by alternative splicing, associates with c-Myc in vivo and inhibits transactivation.

Max (Myc-associated factor X) is a basic helix-loop-helix/leucine zipper protein that has been shown to play a central role in the functional activity of c-Myc as a transcriptional activator. Max potentiates the binding of Myc-Max heterodimers through its basic region to its specific E-box Myc site (EMS), enabling c-Myc to transactivate effectively. In addition to the alternatively spliced exon a, several naturally occurring forms of alternatively spliced max mRNAs have been reported, but variant protein products from these transcripts have not been detected. Using Western blot (immunoblot) and immunoprecipitation analysis, we have identified a variant form of Max protein (16 to 17 kDa), termed dMax, in detergent nuclear extracts of murine B-lymphoma cells, normal B lymphocytes, and NIH 3T3 fibroblasts. Cloning and sequencing revealed that dMax contains a deletion spanning the basic region and helix 1 and the loop of the helix-loop-helix region, presumably as a result of alternative splicing of max RNA. S1 nuclease analysis confirmed the presence of the mRNA for dMax in cells. The dMax protein, prepared via in vitro transcription and translation, associated with bacterially synthesized Myc-glutathione S-transferase. Coimmunoprecipitation of dMax and c-Myc indicated their intracellular association. In vitro-synthesized dMax failed to bind EMS DNA, presumably because of the absence of the basic region. Coexpression of dMax inhibited EMS-mediated transactivation by c-Myc. Thus dMax, which can interact with c-Myc, appears to function as a dominant negative regulator, providing an additional level of regulation to the transactivation potential of c-Myc.

3T3 Cells↗

PubMed1999

Regulation of AUF1 expression via conserved alternatively spliced elements in the 3' untranslated region.

The A+U-rich RNA-binding factor AUF1 exhibits characteristics of a trans-acting factor contributing to the rapid turnover of many cellular mRNAs. Structural mapping of the AUF1 gene and its transcribed mRNA has revealed alternative splicing events within the 3' untranslated region (3'-UTR). In K562 erythroleukemia cells, we have identified four alternatively spliced AUF1 3'-UTR variants, including a population of AUF1 mRNA containing a highly conserved 107-nucleotide (nt) 3'-UTR exon (exon 9) and the adjacent downstream intron (intron 9). Functional analyses using luciferase-AUF1 3'-UTR chimeric transcripts demonstrated that the presence of either a spliceable or an unspliceable intron 9 in the 3'-UTR repressed luciferase expression in cis, indicating that intron 9 sequences may down-regulate gene expression by two distinct mechanisms. In the case of the unspliceable intron, repression of luciferase expression likely involved two AUF1-binding sequences, since luciferase expression was increased by deletion of these sites. However, inclusion of the spliceable intron in the luciferase 3'-UTR down-regulated expression independent of the AUF1-binding sequences. This is likely due to nonsense-mediated mRNA decay (NMD) owing to the generation of exon-exon junctions more than 50 nt downstream of the luciferase termination codon. AUF1 mRNA splice variants generated by selective excision of intron 9 are thus also likely to be subject to NMD since intron 9 is always positioned >137 nt downstream of the stop codon. The distribution of alternatively spliced AUF1 transcripts in K562 cells is consistent with this model of regulated AUF1 expression.

Alternative Splicing↗

PubMed2003

Differential regulation of transmitter release by alternatively spliced forms of synaptotagmin I.

We discovered a novel alternatively spliced form of synaptotagmin I (Syt I). This splicing event is conserved over evolution and, in Aplysia, results in a two amino acid insert in the juxtamembrane domain of Syt I (Syt IVQ). Both Syt I and Syt IVQ are localized to synaptic vesicles; however, we also observed punctae that contained one or the other spliced products. Both Syt I and Syt IVQ are phosphorylated at the adjacent PKC site. Overexpression of Syt IVQ, but not of Syt I, in Aplysia neurons blocked the ability of serotonin to reverse synaptic depression. This effect is upstream of PKC activation, because neither Syt IVQ nor Syt I blocked the effects of phorbol esters on reversing synaptic depression or the effects of serotonin on facilitating nondepressed synapses. Our results demonstrate a physiological role for splicing in the juxtamembrane domain of Syt I.

Alternative Splicing↗