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PubMed1993

Functional properties of alternatively spliced forms of the Drosophila PS2 integrin alpha subunit.

The Drosophila alpha PS2 protein is encoded by two alternatively spliced transcripts. The respective alpha PS2 proteins differ by the presence of 25 amino acids in the alpha PS2(C) protein, not found in the alpha PS2(m8) subunit, in a region thought to be important for ligand binding. We examined the functional properties of Drosophila S2 cells transformed with genes expressing either of these proteins, in association with a beta PS subunit. Both PS2 integrins support cell spreading on vertebrate vitronectin or, to a lesser extent, on fibronectin. Interestingly, the PS2(C) form promotes spreading more efficiently on vitronectin than does the PS2(m8) form, with an opposite relative efficiency seen for fibronectin. Also, the two forms of PS2 show different requirements for divalent cations in order to mediate efficient cell spreading. These divalent cations are not required to maintain the association of alpha and beta subunits. Spreading of both cell types is similarly RGD sensitive, and both PS2 integrins appear to associate with the actin cytoskeleton. To our knowledge, this represents the first demonstration of functional differences in integrin subunits resulting from splicing variation to generate different extracellular, ligand binding domains.

Alternative Splicing↗

PubMed2006

Alternative transcripts of rat slc19a1: defective sorting and inefficient expression of alternative splicing products--an addendum.

Recently, we have reported about the existence and cloning of alternative transcripts of the solute carrier 19a1 in rat liver and kidney, and have explained their origin from tissue specific promoters and by alternative splicing [DNA Seq. 2005, 16(1), 1-6]. The variant open reading frames (ORFs) of these transcripts were now expressed as fusion proteins with an N-terminal GFP. Transfection of HPCT-1E3 hepatocytoma cells as well as the MDCK kidney epithelial cell line with the GFP-fusion of the major splicing form containing 12 transmembrane domains (TMD) resulted in a strong fluorescence at the plasma membrane, which was stable over at least 3 days. Expression of GFP alone gave a comparable overall fluorescence intensity, although, staining was distributed evenly throughout the cells. Fusions of GFP with the shorter ORFs of all alternative slc19a1 transcripts containing 6-7 predicted TMD were expressed less efficiently and not sorted to the plasma membrane. Instead, these proteins accumulated in intracellular granules, and were, apparently, degraded. Hence, it is unlikely that these minor splicing forms are directly involved in solute transport across the plasma membrane.

Alternative Splicing↗

PubMed1989

Essential large transcripts of the maize Spm transposable element are generated by alternative splicing.

We used in vitro mutagenesis and cDNA cloning to identify new Suppressor-mutator (Spm) transposable element genes. Frameshift mutations in the ORFs of the tnpA gene's first intron markedly reduce Spm activity in transgenic tobacco, indicating that intron sequences encode essential gene products. Evidence is given that Spm encodes large alternatively spliced transcripts, designated tnpB (4.9 kb), tnpC (5.7 kb), and tnpD (5.8 kb), comprising all of the tnpA exons, most of the tnpA intron 1 ORF1 sequence, and either none, part, or all of the intron 1 ORF2 sequence. Two alternative splice donor sites were identified at the end of exon 1, and the structure of the different exon 1 sequences suggests that Spm employs a novel mode of translational regulation.

DNA↗

PubMed1984

A single rat fibronectin gene generates three different mRNAs by alternative splicing of a complex exon.

Three fibronectin mRNAs exist in rat liver, differing by the presence or absence of segments of 285 or 360 bases at a point within the coding region. We previously proposed that the three mRNAs are encoded by a single gene and arise via alternative splicing of a common transcript. In order to test this hypothesis, we have isolated clones spanning approximately half of the fibronectin gene from a Fisher rat genomic library; blot hybridization analyses reveal the presence of only one fibronectin gene in the haploid rat genome. We determined the sequence of a portion (1221 nucleotides) of this gene. This sequence shows clearly that the three fibronectin mRNAs encoded by this gene are generated by a pattern of alternative splicing in which one 5' splice site can be paired with any one of three 3' splice sites, one at the beginning of, and two within, a single complex exon.

Amino Acid Sequence↗

PubMed1992

Alternative splicing of micro-exons creates multiple forms of the insect cell adhesion molecule fasciclin I.

Fasciclin I is a homophilic cell adhesion molecule in insects that is dynamically expressed on a subset of axon pathways in the embryonic nervous system, and on a variety of other cells and tissues during development. The fasciclin I protein consists of four homologous 150 amino acid domains. In this article, we describe the complete sequence of the Drosophila fasciclin I (fasI) gene. The gene consists of 15 exons and is distributed over 14 kilobases of DNA. We examine the structure and temporal expression pattern of multiple fasciclin I mRNAs that differ in the lengths of their 3' untranslated regions. We also show that a highly conserved sequence at the end of the second domain can be altered by the addition of three or six amino acids that are encoded by two alternatively spliced 9 base pair (bp) micro-exons. In grasshopper fasciclin I mRNAs, there are 9 bp and 6 bp insertions at the same position. The first of these insertions is identical in sequence to the first fly micro-exon. The grasshopper insertions are not found together in the same mRNA, so grasshopper fasciclin I species differ by the addition of three or two extra amino acids to the second domain. The alternatively spliced mRNAs are differentially expressed during embryogenesis, and all three of them are present in nerve cord preparations. We suggest that the amino acids inserted by alternative micro-exon splicing may alter the binding specificity of fasciclin I.

Animals↗

PubMed1989

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5'-disrupted myb loci.

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.

Amino Acid Sequence↗

PubMed1991

Focal adhesion integrity is downregulated by the alternatively spliced domain of human tenascin.

Tenascin, together with thrombospondin and SPARC, form a family of matrix proteins that, when added to bovine aortic endothelial cells, caused a dose-dependent reduction in the number of focal adhesion-positive cells to approximately 50% of albumin-treated controls. For tenascin, a maximum response was obtained with 20-60 micrograms/ml of protein. The reduction in focal adhesions in tenascin-treated spread cells was observed 10 min after addition of the adhesion modulator, reached the maximum by 45 min, and persisted for at least 4 h in the continued presence of tenascin. This effect was fully reversible, was independent of de novo protein synthesis, and was neutralized by a polyclonal antibody to tenascin. Monoclonal antibodies to specific domains of tenascin (mAbs 81C6 and 127) were used to localize the active site to the alternatively spliced segment of tenascin. Furthermore, a recombinant protein corresponding to the alternatively spliced segment (fibronectin type III domains 6-12) was expressed in Escherichia coli and was active in causing loss of focal adhesions, whereas a recombinant form of a domain (domain 3) containing the RGD sequence had no activity. Chondroitin-6-sulfate effectively neutralized tenascin activity, whereas dermatan sulfate and chondroitin-4-sulfate were less active and heparan sulfate and heparin were essentially inactive. Studies suggest that galactosaminoglycans neutralize tenascin activity through interactions with cell surface molecules. Overall, our results demonstrate that tenascin, thrombospondin, and SPARC, acting as soluble ligands, are able to provoke the loss of focal adhesions in well-spread endothelial cells.

Actins↗

PubMed1995

Coding sequence and alternative splicing of the mouse alpha 1(XI) collagen gene (Col11a1).

Several overlapping cDNA clones corresponding to the entire coding sequence of the mouse alpha 1(XI) collagen gene (Col11 a1) were isolated. The conceptual amino acid translation indicated a high degree of sequence identity (93%) with the human alpha 1(XI) chain. The cloning experiments also revealed alternative splicing of the sequence coding for 85 residues located within the acidic region of the amino-globular domain of alpha 1(XI). Analysis of RNA samples from different embryonic tissues suggested that alternative splicing might be confined to tissue destined to become bone.

Amino Acid Sequence↗

PubMed2005

Alternative splicing facilitates internal ribosome entry on the ornithine decarboxylase mRNA.

Ornithine decarboxylase (ODC) is the ratelimiting enzyme in the biosynthesis of polyamines, which are required for optimal cell growth and proliferation. ODC is overexpressed in many tumors and, conversely, its overexpression induces transformation. We have previously reported that ODC mRNA alternative splicing relieves the translation repression normally imposed by a long and structured 5' untranslated region (UTR), and that the ODC 5' UTR contains an internal ribosome entry site (IRES). Here we show that ODC IRES activity is enhanced following inclusion of alternative sequences generated by splicing at cryptic acceptor sites. Furthermore, the alternative ODC IRES is more sensitive to cell-cycledependent changes in the rate of translation. These findings uncover a new biological property of differentially spliced transcripts. This is the first example of alternative splicing that modulates mRNA translation through the cell cycle in a cap-independent manner.

5' Untranslated Regions↗

PubMed1989

A new epidermal growth factor-like domain in the human core protein for the large cartilage-specific proteoglycan. Evidence for alternative splicing of the domain.

A series of cDNA clones for the human core protein of the large cartilage-specific proteoglycan was isolated. Nucleotide sequencing of the clones provided over 2 kilobases of new coding sequences for the human protein. Comparison with published data for cDNA clones covering the same region in rat and chick indicated that domain 8, the lectin-like domain, is highly conserved among species. In contrast, domain 7 is poorly conserved among species. Some of the cDNA clones also contained an additional structural domain between domains 7 and 8 which was not described in the rat or chick sequences. The additional domain of 38 amino acids was highly homologous to epidermal growth factor (EGF)-like sequences seen in other proteins. Because some cDNA clones contained codons for the EGF-like domain and some did not, the results suggested that the EGF-like domain underwent alternative RNA splicing. To confirm alternative splicing of the EGF-like domain, RNA from cartilage cells was used as a template for the polymerase chain reaction. Products of two sizes were obtained. One had the size predicted for mRNA containing the domain and the other had the size predicted for mRNA not containing the domain. Alternative splicing of an EGF-like domain may provide a mechanism of feedback regulation for both the biosynthetic activity and the proliferation of cartilage cells.

Aggrecans↗

PubMed1996

Fibronectin isoform distribution in the mouse. I. The alternatively spliced EIIIB, EIIIA, and V segments show widespread codistribution in the developing mouse embryo.

Fibronectins (FNs) are extracellular matrix glycoproteins that are essential for embryonic development. In order to gain clues to possible developmental roles played by the particular isoforms of FN, we used indirect immunofluorescence microscopy to examine and compare the distributions of the alternatively spliced EIIIB, EIIIA, and V segments, as well as the total pool of FNs, in serial sections from mouse embryos. Antibodies to each of these segments produced staining patterns that colocalized during gastrulation (E7.5) and during early morphogenesis of somites and notochord (E9.5). During the period of continuing organogenesis in the latter half of gestation (E10.5 to E16.5), the antibodies generally continued to produce similar staining patterns localized to epithelial basement membranes, stromal connective tissues, blood vessel walls, and muscles. However, as development proceeded, there was a gradual decline in the intensity of staining for the spliced segments relative to the total pool of FN, with a particularly noticeable decline in staining for EIIIB and EIIIA segments in certain glandular organs, including the liver. A specific reduction in expression of these latter two segments was also evident in the uterus and placenta at early timepoints in gestation. However, the most dramatic difference in the expression of the spliced segments occurred in developing hyaline cartilage, which showed a selective reduction in staining for the EIIIA segment that was evident in the axial skeletal precursors by E12.5 and complete throughout the embryo by E15.5. Our findings suggest that the alternatively spliced EIIIB, EIIIA, and V segments are included in the FN that is required for the morphogenesis of "FN dependent" structures, including somites, notochord, and the vasculature. Conversely, these segments would appear to play divergent, and sometimes exclusive, biological roles in specific tissues such as liver, cartilage, and placenta.

Animals↗

PubMed1993

Co-expression of collagens II and XI and alternative splicing of exon 2 of collagen II in several developing human tissues.

Northern analyses, RNAase protection assays and in situ hybridizations were used to study the expression of the mRNA for the alpha 2 chain of collagen XI and the two different mRNAs generated from the collagen II gene through alternative splicing of exon 2 in several different tissues of 15-19-week-old fetuses. The highest expression levels of procollagen alpha 2(XI) and alpha 1(II) mRNAs were detected in cartilage, but, using long exposure times, Northern hybridization revealed the presence of the approximately 5.3 kb procollagen alpha 1(II) mRNA in most tissues analysed: calvarial and diaphyseal bone, striated and cardiac muscle, skin, brain, lung, kidney, liver, small intestine and colon. Both alternatively spliced forms of the mRNA were present in these tissues. In cartilage, the short form of the procollagen alpha 1(II) mRNA (without exon 2 sequences) was clearly more abundant, whereas in most of the non-cartilaginous tissues the long form was the predominant one. Low levels of procollagen alpha 2(XI) mRNA were also seen in non-cartilaginous tissues: calvarial and diaphyseal bone, kidney, skin, muscle, intestine, liver, brain, and lung. In all the other positive tissues except brain cortex, both collagen II and XI transcripts were observed. The localization of collagen II and XI signals was identical in cartilage, kidney and skin. However, in cartilage the signal with collagen II probe was much higher than that with the collagen alpha 2(XI) probe. In epidermis the situation was reversed. Our results show considerable co-expression and co-localization of procollagen alpha 1(II) and alpha 2(XI) mRNAs in many tissues of developing human fetuses. Since the collagen alpha 1(II) gene also codes for the alpha 3(XI) chain of collagen XI we propose that some, but not all, of the expression of the collagen II gene in non-cartilaginous tissues relates to collagen XI production.

Base Sequence↗

PubMed1991

Type II collagen mRNA containing an alternatively spliced exon predominates in the chick limb prior to chondrogenesis.

A series of cDNA clones corresponding to the 5' end of the chicken type II collagen mRNA were generated using a single-sided polymerase chain reaction technique. Analysis of these cDNAs showed that the second exon of the gene is alternatively spliced such that it is either present or absent in the mRNA. This exon encodes a 70-amino acid cysteine-rich globular domain which is present in the amino-terminal propeptides of alpha 1(I), alpha 1(III), and alpha 2(V) procollagen chains but which was previously thought to be absent from type II procollagen. Analysis of the expression of the two alternatively spliced forms of the chicken type II collagen mRNA showed that the mRNA without the second exon was the predominant form (approximately 90%) in sternal cartilage from 14-day embryos, but in precartilage limb mesenchyme only the form including the second exon was detected. This later form was also present in a number of non-cartilage tissues including embryonic calvaria, skin, heart, skeletal muscle, and brain; no type II collagen mRNA was detected in liver. Studies of developing limbs from progressive embryonic stages suggest that the appearance of the mRNA lacking the second exon is a relatively late event during chondrogenesis.

Amino Acid Sequence↗

PubMed1991

Alternatively spliced mRNA of the pX region of human T lymphotropic virus type I proviral genome.

The pX region of human T lymphotropic virus type I (HTLV-I) is believed to be expressed as a consequence of a 2 step splicing. It is conceivable, however, that a donor site of the 1st splicing and an acceptor site of the 2nd splicing results in the production of an alternatively spliced mRNA which is capable of coding p21X-III. This possibility was examined by amplifying cDNA derived from HTLV-I+ cells between the 5' LTR and pX region. Bands of 2 different sizes were consistently observed. Sequencing of the longer band corresponded to a cDNA derived from a double-spliced pX mRNA as previously reported. The shorter band was derived from a single-spliced mRNA. HTLV-I+ cell lines had both mRNAs to a varying degree. Expression of p40tax and p21X-III seem to be well correlated with a double-spliced and a single-spliced mRNA, respectively.

Base Sequence↗

PubMed1990

Human endothelial cells synthesize, process, and secrete fibronectin molecules bearing an alternatively spliced type III homology (ED1).

Cellular fibronectin (Fn) bearing an alternatively spliced extra type III structural repeat (ED1) is normally present at low concentrations in blood plasma. The source of this material remains uncertain. In this study, primary cultures of human umbilical vein endothelial cells (HUVEC) labeled with 35S-methionine were observed to synthesize Fn monomers both with and without this segment. Monomers isolated from cell lysates with antibodies to the ED1 sequence comigrated in nonreduced sodium dodecyl sulfate polyacrylamide gel electrophoresis with the slower (designated M1), but not the faster (designated M2), of two major monomeric populations that were recognized by antibodies raised to plasma-derived Fn. The differences between M1 and M2 were not due to glycosylation, since they were also observed between species of Fn monomer purified from cells grown in the presence of tunicamycin. M1 and M2 were both observed to incorporate with a similar rate into dimeric Fn, indicating that Fn monomers with and without the ED1 site can dimerize with similar efficiency. Analysis of reduced samples of Fn isolated from cells with anti-ED1 antibodies indicated the presence of both M1-M1 and M1-M2 dimers. In addition to being incorporated into extracellular matrix, ED1 + Fn was observed to be secreted in soluble form into the medium, potentially reflecting intravascular release of this protein by endothelial cells in vivo.

Antibodies, Monoclonal↗

PubMed2006

PGC-1alpha regulates the isoform mRNA ratio of the alternatively spliced thyroid hormone receptor alpha transcript.

Transcripts derived from the thyroid hormone receptor alpha (TRalpha) gene are alternatively spliced resulting in a functional receptor TRalpha1 and a non-T3-binding variant TRalpha2 that can exert a dominant negative effect on the transactivation functions of other TRs. There is evidence that the ratio of TRalpha isoform transcripts can be modulated and here, we investigate whether the PPARgamma co-activator alpha (PGC-1alpha) has an effect on this splicing process. PGC-1alpha was discovered not only as a transcriptional co-activator, but also has certain motifs characteristic of splicing factors. We demonstrate that PGC-1alpha alters the ratio of endogenously expressed TRalpha isoform transcripts in HepG2 cells, by decreasing TRalpha1 mRNA levels twofold. This change in isoform ratio is accompanied by a decrease in 5'-deiodinase expression, whereas no differences were found in TRbeta1 expression. Deletion of the RNA-processing domain of PGC-1alpha abrogated the effect on the TRalpha splicing, whereas expression of only the RNA-processing domain favored TRalpha1 expression. PGC-1alpha showed a similar effect on the splicing of a TRalpha minigene containing only the last four exons and introns of the TRalpha gene. These data suggest that PGC-1alpha is involved in the RNA processing of TRalpha transcripts.

Alternative Splicing↗

PubMed2026

Identification and validation of an alternatively spliced novel isoform of maspin that modulate genes involved in inflammatory and apoptotic pathways.

Maspin regulates cellular adhesion, migration, apoptosis, angiogenesis, and tumor suppression in a tissue and context-dependent manner. Its functional diversity is governed largely thorough extracellular matrix interactions, subcellular localization, and the reactive center loop (RCL), although the structural details are not well understood. To examine whether alternative splicing contributes to this heterogeneity, we analysed the SERPINB5 gene using a computational genomics approach and identified a novel 80 bp coding exon upstream of the first coding exon (E1). The alternatively spliced transcript was validated in human skin and esophagus by semi-nested touchdown PCR, quantitative real-time PCR, and Sanger sequencing. Recombinant B5N displayed a red-shifted fluorescence emission spectrum, indicating a more solvent-exposed conformation, which was supported by molecular dynamics simulations showing greater exposure of the nuclear localization signal (NLS) and the reactive center loop. Enzyme kinetic assays demonstrated concentration-dependent enhancement of tissue plasminogen activator (tPA) activity by both isoforms. In HaCaT cells, wildtype maspin produced stronger antiproliferative and anti-migratory effects, whereas B5N was only mildly antiproliferative. Annexin V/7-AAD staining revealed that wildtype maspin induced higher early apoptosis and cell death, while B5N produced lower overall cell death but a greater proportion of late apoptotic cells. RNA-seq of transfected HaCaT cells identified differentially expressed genes enriched in inflammatory, antiviral, and apoptotic pathways, which was validated by qPCR, and several of these were markedly upregulated in SARS-CoV-2 infected A549 cells. Thus, a novel N-terminally extended maspin isoform with differentially regulated gene profile is identified and validated in this study.

Apoptosis↗

PubMed1997

Cloning and characterization of Kir3.1 (GIRK1) C-terminal alternative splice variants.

Southern blot analysis of RT-PCR products from brain and heart revealed multiple products for a C-terminal region of Kir3.1. Sequencing yielded clones for wild-type Kir3.1 and three Kir3.1 C-terminal alternative splice variants, including a unique alternative exon. Two of these variants encoded truncated Kir3.1 molecules. Tissue distribution and electrophysiological characterization of a single truncated variant, Kir3.1(00) were then examined. Kir3.1 channels are gated by G-protein beta gamma-subunits binding to the C-terminal domain, thus, the truncation of Kir3.1(00) removes a major functional domain. When incorporated into heteromeric channels with other family members (Kir3.1, 3.2 or 3.4) several functional changes were observed: (1) Kir3.1(00) changes G-protein activation of Kir3 channels; (2) Kir3.1(00) is restricted in its ability to assemble with other channel subunits as heteromers; and (3) incorporation of Kir3.1(00) into heteromeric channel complexes alters the kinetics of channel re-activation.

Alternative Splicing↗