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PubMed2000

High incidence of alternatively spliced forms of deoxycytidine kinase in patients with resistant acute myeloid leukemia.

Deficiency of functional deoxycytidine kinase (dCK) is a common characteristic for in vitro resistance to cytarabine (AraC). To investigate whether dCK is also a target for induction of AraC resistance in patients with acute myeloid leukemia (AML), we determined dCK messenger RNA (mRNA) expression in (purified) leukemic blasts and phytohemagglutinin-stimulated T cells (PHA T cells) from patients with chemotherapy-sensitive and chemotherapy-resistant AML. In control samples from healthy donors (PHA T cells and bone marrow), only wild-type dCK complementary DNA (cDNA) was amplified. Also, in (purified) leukemic blasts from patients with sensitive AML, only wild-type dCK cDNAs were observed. These cDNAs coded for active dCK proteins in vitro. However, in 7 of 12 (purified) leukemic blast samples from patients with resistant AML, additional polymerase chain reaction fragments with a deletion of exon 5, exons 3 to 4, exons 3 to 6, or exons 2 to 6 were detected in coexpression with wild-type dCK. Deletion of exons 3 to 6 was also identified in 6 of 12 PHA T cells generated from the patients with resistant AML. The deleted dCK mRNAs were formed by alternative splicing and did code for inactive dCK proteins in vitro. These findings suggest that the presence of inactive, alternatively spliced dCK mRNA transcripts in resistant AML blasts may contribute to the process of AraC resistance in patients with AML. (Blood. 2000;96:1517-1524)

Acute Disease↗

PubMed2001

Molecular and functional characterization of the stress-induced protein (SIP) gene and its two transcripts generated by alternative splicing. SIP induced by stress and promotes cell death.

We have used a quantitative fluorescent cDNA microarray hybridization approach to identify pancreatic genes induced by the cellular stress promoted by acute pancreatitis in the mouse. We report the cloning and characterization of one of them that encodes the stress-induced proteins (SIP). The mouse SIP gene is organized into five exons and expands over approximately 20 kilobase pairs. Exon 4 (38 base pairs) is alternatively spliced to generate two transcripts. Northern blot and in situ hybridization showed that both SIP mRNAs are rapidly and strongly induced in acinar cells of the pancreas with acute pancreatitis. They are also constitutively expressed in several other tissues, although with different ratios. They encode proteins of 18 and 27 kDa (SIP(18) and SIP(27)). SIP(27) is identical to the thymus-expressed acidic protein (TEAP) protein, formerly described as a thymus-specific protein. Expression of the SIP(18) and SIP(27)/EGFP or V5 fusion proteins showed that both are nuclear factors. We monitored SIP expression in NIH3T3 cells submitted to various stress agents. UV stress, base damaging, mutagenic stress, ethanol, heat shock, and oxidative stress induced the concomitant expression of SIP(18) and SIP(27) mRNAs. Finally, transient transfection of SIP(18) and SIP(27) expression plasmids induced death by apoptosis in COS7 cells as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining. In conclusion, the SIP gene is an important element of cellular stress response. It is expressed in many tissues and induced by a variety of stress agents affecting many cellular pathways. SIP generates, by alternative splicing, two nuclear proteins that can promote cell death by apoptosis.

3T3 Cells↗

PubMed1994

Cell surface annexin II is a high affinity receptor for the alternatively spliced segment of tenascin-C.

We have investigated the binding of soluble tenascin-C (TN-C) to several cell lines using a radioligand binding assay. Specific binding was demonstrated to U-251MG human glioma cells and to a line of bovine aortic endothelial cells, but hamster fibroblasts showed no specific binding. Recombinant proteins corresponding to specific domains of TN-C were used to map the binding site(s) in TN-C. The alternatively spliced segment (TNfnA-D) inhibited the binding of native TN-C most strongly, and itself bound to glioma and endothelial cells. Scatchard analysis of TNfnA-D binding indicated 2-5 x 10(5) binding sites per cell, with an apparent 2 nM dissociation constant. The cell surface receptor for TNfnA-D was identified as a 35-kD protein on the basis of blot binding assays and affinity chromatography of membrane extracts on native TN-C and TNfnA-D columns. Protein sequencing indicated that this 35-kD receptor was annexin II. Annexin II is well characterized as a cytoplasmic protein, so it was surprising to find it as a presumably extracellular receptor for TN-C. To confirm that it was the 35-kD receptor, we obtained purified annexin II and demonstrated its binding to TNfnA-D and TN-C at nM concentrations. Antibodies to annexin II prominently stained the external surface of live endothelial cells and blocked the binding of TNfnA-D to the cells. Thus annexin II appears to be a receptor for the alternatively spliced segment of TN-C, and may mediate cellular responses to soluble TN-C in the extracellular matrix.

Alternative Splicing↗

PubMed2007

Modern origin of numerous alternatively spliced human introns from tandem arrays.

Despite the widespread occurrence of spliceosomal introns in the genomes of higher eukaryotes, their origin remains controversial. One model proposes that the duplication of small genomic portions could have provided the boundaries for new introns. If this mechanism has occurred recently, the 5' and 3' boundaries of each resulting intron should display distinctive sequence similarity. Here, we report that the human genome contains an excess of introns with perfect matching sequences at boundaries. One-third of these introns interrupt the protein-coding sequences of known genes. Introns with the best-matching boundaries are invariably found in tandem arrays of direct repeats. Sequence analysis of the arrays indicates that many intron-breeding repeats have disseminated in several genes at different times during human evolution. A comparison with orthologous regions in mouse and chimpanzee suggests a young age for the human introns with the most-similar boundaries. Finally, we show that these human introns are alternatively spliced with exceptionally high frequency. Our study indicates that genomic duplication has been an important mode of intron gain in mammals. The alternative splicing of transcripts containing these intron-breeding repeats may provide the plasticity required for the rapid evolution of new human proteins.

Alternative Splicing↗

PubMed1999

Genomic organization and structure of the 3' region of human MUC3: alternative splicing predicts membrane-bound and soluble forms of the mucin.

The MUC3 gene encodes a large, glycosylated mucin produced by intestinal epithelial cells to form a protective barrier against the external environment. Recently published cDNA sequences for the carboxyl-terminal region of MUC3 polypeptide indicated that rodent Muc3 possesses two epidermal growth factor (EGF)-like domains, and putative transmembrane and cytoplasmic domains, whereas the sequence of human MUC3 predicted termination after the first EGF-like domain. Here we describe the complete genomic sequence encompassing the carboxyl terminal region of human MUC3, revealing the boundaries of 11 exons. RT-PCR and cDNA library cloning experiments indicate that the gene is alternatively spliced, yielding a major membrane-bound form as well as multiple soluble forms. Thus, this work indicates that both membrane-bound and soluble MUC3 mucin proteins are produced by alternative splicing of a single gene. A potentially important polymorphism involving a Tyr residue with a proposed role in signalling is described as well.

3' Untranslated Regions↗

PubMed1993

Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain.

The NMDA (N-methyl D-aspartate) receptors in the brain play a critical role in synaptic plasticity, synaptogenesis and excitotoxicity. Molecular cloning has demonstrated that NMDA receptors consist of several homologous subunits (NMDAR1, 2A-2D). A variety of studies have suggested that protein phosphorylation of NMDA receptors may regulate their function and play a role in many forms of synaptic plasticity such as long-term potentiation. We have examined the phosphorylation of the NMDA receptor subunit NMDAR1 (NR1) by protein kinase C (PKC) in cells transiently expressing recombinant NR1 and in primary cultures of cortical neurons. PKC phosphorylation occurs on several distinct sites on the NR1 subunit. Most of these sites are contained within a single alternatively spliced exon in the C-terminal domain, which has previously been proposed to be on the extracellular side of the membrane. These results demonstrate that alternative splicing of the NR1 messenger RNA regulates its phosphorylation by PKC, and that mRNA splicing is a novel mechanism for regulating the sensitivity of glutamate receptors to protein phosphorylation. These results also provide evidence that the C-terminal domain of the NR1 protein is located intracellularly, suggesting that the proposed transmembrane topology model for glutamate receptors may be incorrect.

Alternative Splicing↗

PubMed2003

Tumor-related alternatively spliced Rac1b is not regulated by Rho-GDP dissociation inhibitors and exhibits selective downstream signaling.

Rac1 is a member of the Rho family of small GTPases, which control signaling pathways that regulate actin cytoskeletal dynamics and gene transcription. Rac1 is activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins. In addition, Rho-GDP dissociation inhibitors (Rho-GDIs) can inhibit Rac1 by sequestering it in the cytoplasm. We have found previously that colorectal tumors express an alternatively spliced variant, Rac1b, containing 19 additional amino acids following the switch II region. Here we characterized the regulation and downstream signaling of Rac1b. Although little Rac1b protein is expressed in cells, the amount of activated Rac1b protein often exceeds that of activated Rac1, suggesting that Rac1b contributes significantly to the downstream signaling of Rac in cells. The regulation of both Rac1 and Rac1b activities is dependent on guanine nucleotide exchange factors and GTPase-activating proteins, but the difference in their activation is mainly determined by the inability of Rac1b to interact with Rho-GDI. As a consequence, most Rac1b remains bound to the plasma membrane and is not sequestered by Rho-GDI in the cytoplasm. Unlike Rac1, activated Rac1b is unable to induce lamellipodia formation and is unable to bind and activate p21-activated protein kinase nor activate the downstream protein kinase JNK. However, both Rac1 and Rac1b are able to activate NFkappaB to the same extent. These data suggest that alternative splicing of Rac1 leads to a highly active Rac variant that differs in regulation and downstream signaling.

Alternative Splicing↗

PubMedDate not supplied

Quantitative measurement of alternatively spliced amyloid precursor protein mRNA expression in Alzheimer's disease and normal brain by S1 nuclease protection analysis.

We have used an S1 nuclease protection strategy to measure alternatively spliced amyloid precursor protein (APP) mRNAs associated with Alzheimer's disease (AD) to determine whether the expression of either one or more of the transcripts correlate with observed amyloid plaque pathology. Comparison of AD with normal cortex reveals that increasing plaque density parallels an increase in the fraction of APP-695 and a corresponding decrease in APP-770 and 751 mRNA fractions. A specific increase of APP-695, the protease inhibitor-lacking APP RNA form, in those brain regions most involved with amyloid plaque formation, suggests that an imbalance in the protease inhibitor is potentially significant in the disease. These data are consistent with cellular/tissue region-specific regulation of alternative splicing accounting for AD-related changes in the expression of APP mRNA forms.

Adult↗

PubMedDate not supplied

Identification of calretinin and the alternatively spliced form calretinin-22k in primary pleural mesotheliomas and in their metastases.

BACKGROUND: Antibodies against calretinin represent an established, powerful and reliable immunohistochemical marker in the differential diagnosis between mesothelioma and adenocarcinomas. However, in studies published so far, the exact molecular identity of the immunoreactive protein(s) detected in mesothelioma sections has not yet been determined. MATERIALS AND METHODS: Tissue biopsies from ten mesothelioma samples, primary and metastatic, were analyzed by Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR), in addition to immunohistochemical staining. RESULTS: Besides the selective positive immunoreaction in all 10 samples, calretinin was identified by Western blot analysis and by identification of its mRNA by RT-PCR. In addition, we identified the alternatively spliced form calretinin-22k and the corresponding mRNA, previously detected only in neoplastic cells in colon carcinomas and derived cell lines. CONCLUSION: Our findings demonstrate that the immunoreactivity observed in the mesotheliomas and their metastases investigated in this report is due to the concomitant presence of calretinin and the alternatively spliced form calretinin-22k.

Adult↗

PubMed1995

Developmental expression of p107 mRNA and evidence for alternative splicing of the p107 (RBL1) gene product.

Expression of p107, a protein with homology to the retinoblastoma tumor suppressor (pRB), was monitored during murine development. Northern blot tissue surveys identified two transcripts of 4.9 and 2.4 kb that hybridized to a p107 cDNA clone. Expression of both transcripts was detected in fetal tissues, with particularly high levels in the liver and heart. In contrast, p107 transcripts were markedly decreased in most adult tissues examined. Molecular cloning analyses revealed that the 4.9- and 2.4-kb transcripts encoded proteins with deduced molecular masses of 119 and 68 kDa, respectively. Genetic mapping studies suggested that the two p107 transcripts arose by alternative splicing of a common precursor. The protein encoded by the 2.4-kb transcript lacks the spacer and B motif of the "pocket domain," a region of homology between p107 and pRB that is required for binding to cell cycle regulatory proteins. Structural modifications resulting from alternative splicing may thus confer functional diversity upon the 119- and 68-kDa proteins.

Alternative Splicing↗

PubMed1996

A large variety of alternatively spliced and differentially expressed mRNAs are encoded by the human acute myeloid leukemia gene AML1.

The human chromosome 21 acute myeloid leukemia gene AML1 is frequently rearranged in the leukemia-associated translocations t(8;21) and t(3;21), generating fused proteins containing the amino-terminal part of AML1. In normal blood cells, five size classes (2-8 kb) of AML1 mRNAs have been previously observed. We isolated seven cDNAs corresponding to various AML1 mRNAs. Sequencing revealed that their size differences were mainly due to alternatively spliced 5' and 3' untranslated regions, some of which were vast, exceeding 1.5 kb (5') and 4.3 kb (3'). These untranslated regions contain sequences known to control mRNA translation and stability and seem to modulate AML1 mRNA stability. Further heterogeneity was found in the coding region due to the presence of alternatively spliced stop codon-containing exons. The latter led to production of polypeptides that were smaller than the full-length AML1 protein; they lacked the trans-activation domains but maintained DNA binding and heterodimerization ability. The size of these truncated products was similar to the AML1 segment in the fused t(8;21) and t(3;21) proteins. In thymus, only one mRNA species of 6 kb was detected. Using in situ hybridization, we showed that its expression was confined to the cortical region of the organ. The 6-kb mRNA was also prominent in cultured peripheral blood T cells, and its expression was markedly reduced upon mitogenic activation by phorbol myristate acetate (TPA) plus concanavalin A (ConA). These results and the presence of multiple coding regions flanked by long complex untranslated regions, suggest that AML1 expression is regulated at different levels by several control mechanisms generating the large variety of mRNAs and protein products.

Alternative Splicing↗

PubMed2003

Molecular cloning and characterization of gilthead sea bream (Sparus aurata) growth hormone receptor (GHR). Assessment of alternative splicing.

The full-length growth hormone receptor (GHR) of gilthead sea bream (Sparus aurata) was cloned and sequenced by RT-PCR and rapid amplification of 5'and 3'ends. The open reading frame codes for a mature 609 amino acid protein with a hydrophobic transmembrane region and all the characteristic motifs of GHRs. Sequence analysis revealed a 96 and 76% of amino acid identity with black sea bream (Acanthopagrus schlegeli) and turbot (Scophthalmus maximus) GHRs, respectively, but this amino acid identity decreases up to 52% for goldfish (Carassius auratus) GHR. By means of real-time PCR assays, concurrent changes in the hepatic expression of GHRs and insulin-like growth factor-I (IGF-I) was evidenced. Moreover, their regulation occurred in conjunction with the summer spurt of growth rates and circulating levels of GH and IGF-I. Search of alternative splicing was carried out exhaustively for gilthead sea bream GHR, but Northern blot and 3' RACE failed to demonstrate the occurrence of short alternative messengers. Besides, RT-PCR screening did not reveal deletions or insertions that could lead to alternative reading frames. In agreement with this, cross-linking assays only evidenced two protein bands that match well with the size of glycosylated and non-glycosylated forms of the full-length GHR. If so, it appears that alternative splicing at the 3'end does not occur in gilthead sea bream, although different messengers for truncated or longer GHR variants already exist in turbot and black sea bream, respectively. The physiological relevance of this finding remains unclear, but perhaps it points out large inter-species differences in the heterogeneity of the GHR population.

Alternative Splicing↗

PubMed2000

A novel subgenomic murine leukemia virus RNA transcript results from alternative splicing.

Here we show the existence of a novel subgenomic 4.4-kb RNA in cells infected with the prototypic replication-competent Friend or Moloney murine leukemia viruses (MuLV). This RNA derives by splicing from an alternative donor site (SD') within the capsid-coding region to the canonical envelope splice acceptor site. The position and the sequence of SD' was highly conserved among mammalian type C and D oncoviruses. Point mutations used to inactivate SD' without changing the capsid-coding ability affected viral RNA splicing and reduced viral replication in infected cells.

Alternative Splicing↗

PubMedDate not supplied

[Alternative splicing of pre-mRNA encoding the Musca domestica latrophilin-like protein(LLP): primary structures of four spliced forms of mRNA and their protein products].

An internal DNA fragment (approximately 2000 bp) homologous to the conserved regions of genes encoding latrophilin-like proteins (LLPs) was obtained by the PCR technique using degenerate primers to these gene regions. The gene-specific primers were synthesized based on the results of sequencing of the isolated fragment, and all overlapping cDNA fragments of the llp gene encoding the Musca domestica LLP were obtained by the rapid amplification of cDNA 5'- and 3'-ends (5'- and 3'-RACE). Four alternatively spliced mRNAs were found while sequencing the obtained cDNA fragments. Two long mRNAs (approximately 6000 nt) differ in the structures of both the sites encoding signal peptides and 5'-terminal untranslated regions. They encode large proteins (approximately 1800 aa), whose domain organization is similar to that of mammalian latrophilins. Each deduced protein contains a domain with seven transmembrane regions followed by an extended cytoplasmic C-terminal domain. Two other mRNA forms are derived from these long mRNAs; they encode proteins severly truncated at their C-termini (approximately 900 aa). They are composed of only three transmembrane regions and a short unique cytoplasmic C-terminal domain (23 aa). The limitations and drawbacks of the existing 3'-RACE techniques found during study of the long alternatively spliced cDNAs are analyzed, and ways for overcoming these difficulties are proposed.

Alternative Splicing↗

PubMed1998

Multiple novel transcripts for apolipoprotein(a)-related gene II generated by alternative splicing in tissue- and cell type-specific manners.

Various Kringle 4 (K4) sequences were identified in human genomic clones and genomic DNAs amplified by PCR. These K4s were homologous to those found in the apo(a) gene and thus termed apo(a)-related genes (ARGs). The same sequences were obtained when human peripheral leukocytes were analyzed by reverse transcription-PCR in order to study the expression mode of the ARGs. It was of note that multiple transcripts with three optional exons for an ARG (ARGII) were detected in leukocytes, indicating that they were generated by alternative splicing. All these transcripts possessed the first half of the second K4 sequence, which had been reported to be skipped. The variant products of ARGII are expected to contain an additional region of either 44, 66, or 100 unique amino acids at the C-terminus of a single K4 unit. When normal human tissues and cultured tumor cells were analyzed, the multiple ARGII transcripts were detected at varying levels. The presence of the cellular state-specific alternative splicing machinery may provide not only redundancy but also diversity in the structure/function of ARGII.

Alternative Splicing↗

PubMed2004

Alternative splicing generates multiple SMRT transcripts encoding conserved repressor domains linked to variable transcription factor interaction domains.

Silencing mediator for retinoid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor protein (NCoR) are corepressors that interact with a range of transcription factors. They both consist of N-terminal repressor domains that associate with histone deacetylases and C-terminal interaction domains (IDs) that contain CoRNR box motifs. These motifs mediate the interaction between corepressors and nuclear receptors (NRs), such as the retinoid and thyroid hormone receptors. However, whilst NCoR produces a single transcript during Xenopus development, xSMRT is subject to alternative splicing at four sites in the 3' part of the transcript, the region encoding the C-terminal IDs. Although this provides the potential to produce 16 different transcripts, only five isoforms are found in early embryos. The sites of alternative splicing predict that the resultant isoforms will differ in their ability to interact with NRs, as one site varies the number of CoRNR boxes, the second site changes the sequence flanking CoRNR box-1 and the other sites delete amino acid residues between CoRNR boxes 1 and 2 and so alter the critical spacing between these motifs. SMRT and NCoR therefore represent paralogues in which one form, SMRT, has evolved the ability to generate multiple isoforms whereas the other, NCoR, is invariant in Xenopus development.

Alternative Splicing↗

PubMed1997

Use of an alternate splice donor site in the human GAP gene is responsible for synthesis of the p100 isoform.

GAP (GTPase-activating protein), a negative regulator of the receptor tyrosine kinase signal transduction pathway, exists as two isoforms: a ubiquitous, p120 form and a primate placenta-specific p100 form lacking the N-terminal hydrophobic domain. The cDNA species encoding p120 and p100 GAP are identical except that p100 GAP cDNA contains a 65-bp insert not present in p120 cDNA. The purpose of this study was to locate the 65-bp insert in the genomic GAP sequence, thereby determining the mechanism by which alternate splicing produces the two mRNA species. It was found that the 65-bp insert is located just 3' to the sequence encoding the hydrophobic domain, indicating that the p100 form of GAP results from utilization of an alternate splice donor site. In addition, the sequence encoding the hydrophobic domain was found to be contained within a single large exon. The intron separating this exon from the exon encoding the 5'-portion of the SH2-N domain was determined to be at least 40 kb in length. Finally, it was found that the sequence encoding the SH2-N domain contains an intron 1006 bp long, and the sequence of this intron has been deduced. It is anticipated that the data presented in this paper will provide the basis for elucidating RNA processing mechanisms responsible for preferential expression of p100 GAP in the human placenta.

Alternative Splicing↗

PubMed2000

Stretch-induced alternative splicing of serum response factor promotes bronchial myogenesis and is defective in lung hypoplasia.

Smooth muscle (SM) develops only in organs and sites that sustain mechanical tensions. Therefore, we determined the role of stretch in mouse and human bronchial myogenesis. Sustained stretch induced expression of SM proteins in undifferentiated mesenchymal cells and accelerated the differentiation of cells undergoing myogenesis. Moreover, bronchial myogenesis was entirely controlled in lung organ cultures by the airway intraluminal pressure. Serum response factor (SRF) is a transcription factor critical for the induction of muscle-specific gene expression. Recently, a SRF-truncated isoform produced by alternative splicing of exon 5 has been identified (SRFDelta5). Here we show that undifferentiated mesenchymal cells synthesize both SRF and SRFDelta5 but that SRFDelta5 synthesis is suppressed during bronchial myogenesis in favor of increased SRF production. Stretch induces the same change in SRF alternative splicing, and its myogenic effect is abrogated by overexpressing SRFDelta5. Furthermore, human hypoplastic lungs related to conditions that hinder cell stretching continue to synthesize SRFDelta5 and show a marked decrease in bronchial and interstitial SM cells and their ECM product, tropoelastin. Taken together, our findings indicate that stretch plays a critical role in SM myogenesis and suggest that its decrease precludes normal bronchial muscle development.

Alternative Splicing↗