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Results for “Alternative Splicing”

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PubMed1995

Expression of alternatively spliced fibronectin variants during remodeling in proliferative glomerulonephritis.

Fibronectin (Fn) plays an important role in tissue remodeling during embryogenesis, wound repair, and vascular disease, and is thought to regulate cellular processes such as cell adhesion, migration, proliferation, and differentiation through specialized domains within the molecule. In addition, Fn can be alternatively spliced at three regions: extradomains EIIIA, EIIIB, and a variable segment V, potentially giving rise to functionally distinct variants of the molecule. We have previously shown a sequential expression of cellular Fn first by platelets, followed by macrophages, then mesangial cells in habu snake venom-induced proliferative glomerulonephritis (Am J Pathol 145: 585-597, 1994). These studies examined the cellular sources and glomerular localization of Fn in general but did not distinguish between the various alternatively spliced isoforms. In this study, we examine by in situ hybridization and immunohistochemistry the temporal expression and cellular sources of EIIIA, EIIIB, and V in a model of proliferation glomerulonephritis that has cell migration, proliferation, and extracellular matrix synthesis as features of tissue remodeling. Macrophages were the first cells to express Fn mRNA showing an EIIIA+, EIIIB-, and V95+ pattern beginning at 8 hours after habu snake venom injection. Migrating mesangial cells at the margins of early lesions (8 and 24 hours) did not overexpress mRNA encoding these Fn variants, but immunofluorescence microscopy revealed V95 and EIIIA protein at the margins of lesions. EIIIB was absent in lesions at this time. At 48 hours and peaking at 72 hours after habu snake venom injection, mesangial cells in central aspects of glomerular lesions expressed abundant mRNA and protein for V95 and EIIIA. EIIIB mRNA and protein was slight in the mesangium at these times. Parietal epithelial cells, particularly adjacent to glomerular lesions, also expressed abundant mRNA and protein for all three variants throughout the course of the disease, beginning at 24 hours after habu snake venom injection. Expression of mRNA and protein for all three isoforms declined by 2 weeks after habu snake venom injection. These studies show that migrating mesangial cells do not require their own synthesis of Fn and suggest that they might rely on exogenous sources of Fn, particularly V95+ and EIIIA+ forms. Commencement of enhanced expression of EIIIA and EIIIB mRNA and protein by resident glomerular cells coincided with the temporal course of cell proliferation, acquisition of alpha-smooth muscle cell actin phenotype, and matrix synthesis, suggesting that Fn isoforms have specific functions during the course of glomerular remodeling.

Alternative Splicing↗

PubMed1994

Control of polyadenylation and alternative splicing of transcripts from adjacent genes in a procyclin expression site: a dual role for polypyrimidine tracts in trypanosomes?

The procyclin-associated genes (PAGs) of Trypanosoma brucei are located downstream of tandemly repeated procyclin genes and belong to the same alpha-amanitin-resistant polycistronic transcription units. In procyclic form trypanosomes the PAG 1 pre-mRNA is alternatively spliced to give rise to three transcripts of 2.7 kb, 1.8 kb and 1.3 kb. The two larger transcripts contain additional short open reading frames (ORFs) upstream of the major ORF. Trans-splicing to generate these transcripts occurs downstream of three different polypyrimidine tracts. A minor population of procyclin mRNAs is also generated by alternative splicing at a polypyrimidine tract that begins 524 bp upstream of the major splice acceptor site of the procyclin beta-gene. The same polypyrimidine tract is also required for accurate polyadenylation of mRNAs from the upstream procyclin alpha-gene (1). Alternatively polyadenylated forms of PAG 1 mRNAs can also be detected. All polyadenylation sites are found at a similar distance upstream of splice-acceptor sites, in each case with a polypyrimidine tract between them. Our results point to a dual role for polypyrimidine tracts in the maturation of trypanosome mRNAs.

Alternative Splicing↗

PubMed1989

Probing molecular polymorphism of fibronectins with antibodies directed to the alternatively spliced peptide segments.

Molecular heterogeneity of fibronectins (FNs) isolated from plasma, cultured fibroblasts, and placenta was studied with site-specific antibodies recognizing alternatively spliced peptide segments, termed ED-A and IIICS/delta 2. The antibodies were raised in rabbits by immunization with synthetic peptides. Neither the ED-A nor the IIICS/delta 2 extra peptide segment was present in the major subunits of plasma FN, although a minor subunit contained the latter extra segment. Cellular FN consisted of at least four subunits differing in size of the fragments generated by cleavage of the C-terminal region with cathepsin D. These fragments were distinct from each other in the reactivity with anti-ED-A and anti-IIICS/delta 2 antibodies, suggesting that all combinations of the presence or absence of the extra segments were produced by cultured fibroblasts. Placental FN was more heterogeneous than plasma and cellular FNs, consisting of five, or probably more, subunits. Among these, the two smaller subunits appeared to be closely similar to the major subunits of plasma FN, whereas the other subunits were more related to those of cellular FN in the size of cathepsin D cleaved C-terminal fragments and in the reactivity with anti-peptide antibodies. These results, taken together, indicate that the FNs produced by different tissues or cell types are distinct from each other in the number and types of subunits, which are partly, if not all, defined by alternative splicing at the ED-A and IIICS regions.

Antibodies↗

PubMed2002

Regulation of alternative splicing of alpha-actinin transcript by Bruno-like proteins.

BACKGROUND: The Bruno-like or CELF proteins, such as mammalian CUGBP1 and Etr-3, Xenopus EDEN-BP, and Drosophila Bruno (Bru), are regulators of gene expression at the post-transcriptional level, and contain three RNA-recognition motifs (RRMs). It has been shown that mammalian CUGBP1 and Etr-3 regulate alternative splicing of cardiac troponin T pre-mRNA via binding to CUG-triplet repeats. RESULTS: Using in vitro selection and UV-crosslinking experiments, we found that zebrafish Bruno-like proteins bound to repeat elements of uridine and purine (termed UREs). It is known that non-muscle (NM) and smooth muscle (SM) exons of the rat alpha-actinin gene are used in a mutually exclusive manner. Transfection experiments in mammalian cells showed that zebrafish Brul and Etr-3 induced the muscle-specific splicing of rat alpha-actinin pre-mRNA via binding to the URE at the branch point upstream of the NM exon. In contrast, zebrafish Etr-1 promoted skipping of both the NM and SM exons in a manner which was not dependent on URE-binding. CONCLUSIONS: Our results showed that Bruno-like proteins bind to UREs and regulate the alternative splicing of alpha-actinin pre-mRNA. Members of the Bruno family play multiple roles in splicing regulation.

Actinin↗

PubMed2001

Alternative splicing in intracellular loop connecting domains II and III of the alpha 1 subunit of Cav1.2 Ca2+ channels predicts two-domain polypeptides with unique C-terminal tails.

Novel splice variants of the alpha(1) subunit of the Ca(v)1.2 voltage-gated Ca(2+) channel were identified that predicted two truncated forms of the alpha(1) subunit comprising domains I and II generated by alternative splicing in the intracellular loop region linking domains II and III. In rabbit heart splice variant 1 (RH-1), exon 19 was deleted, which resulted in a reading frameshift of exon 20 with a premature termination codon and a novel 19-amino acid carboxyl-terminal tail. In the RH-2 variant, exons 17 and 18 were deleted, leading to a reading frameshift of exons 19 and 20 with a premature stop codon and a novel 62-amino acid carboxyl-terminal tail. RNase protection assays with RH-1 and RH-2 cRNA probes confirmed the expression in cardiac and neuronal tissue but not skeletal muscle. The deduced amino acid sequence from full-length cDNAs encoding the two variants predicted polypeptides of 99.0 and 99.2 kDa, which constituted domains I and II of the alpha(1) subunit of the Ca(v)1.2 channel. Antipeptide antibodies directed to sequences in the second intracellular loop between domains II and III identified the 240-kDa Ca(v)1.2 subunit in sarcolemmal and heavy sarcoplasmic reticulum (HSR) membranes and a 99-kDa polypeptide in the HSR. An antipeptide antibody raised against unique sequences in the RH-2 variant also identified a 99-kDa polypeptide in the HSR. These data reveal the expression of additional Ca(2+) channel structural units generated by alternative splicing of the Ca(v)1.2 gene.

Alternative Splicing↗

PubMed2005

Evolutionarily conserved coupling of transcription and alternative splicing in the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes.

Recent studies have shown that transcription and alternative splicing can be mechanistically coupled. In the EPB41 (protein 4.1R) and EPB41L3 (protein 4.1B) genes, we showed previously that promoter/alternative first exon choice is coupled to downstream splicing events in exon 2. Here we demonstrate that this coupling is conserved among several vertebrate classes from fish to mammals. The EPB41 and EPB41L3 genes from fish, bird, amphibian, and mammal genomes exhibit shared features including alternative first exons and differential splice acceptors in exon 2. In all cases, the 5'-most exon (exon 1A) splices exclusively to a weaker internal acceptor site in exon 2, skipping a fragment designated as exon 2'. Conversely, alternative first exons 1B and 1C always splice to the stronger first acceptor site, retaining exon 2'. These correlations are independent of cell type or species of origin. Since exon 2' contains a translation initiation site, splice variants generate protein isoforms with distinct N-termini. We propose that these genes represent a physiologically relevant model system for mechanistic analysis of transcription-coupled alternative splicing.

Alternative Splicing↗

PubMed2006

Porcine T-cell receptor beta-chain: a genomic sequence covering Dbeta1.1 to Cbeta2 gene segments and the diversity of cDNA expressed in piglets including novel alternative splicing products.

Porcine TCRbeta-chain cDNA clones were isolated from thymic and peripheral blood lymphocytes of piglets. Using these nucleotide sequences, a genomic 18kbp sequence stretch covering Dbeta1 to Cbeta2 gene segments was identified, which revealed that the porcine TCRbeta-chain locus consists of two sets of Dbeta-Jbeta-Cbeta gene groups with each set having a Dbeta gene segment, seven Jbeta gene segments and a down stream Cbeta gene segment composed of four exons. This structure is consistent with other known mammalian TCRbeta-chain loci. With this genomic information, TCRbeta-chain clones from cDNA libraries were analyzed. Sixteen Vbeta gene segments were obtained accompanied by either Dbeta1 or Dbeta2 and by one of the nine Jbeta gene segments. Five different Cbeta cDNA sequences were obtained including four types of Cbeta1 sequences and one type of Cbeta2 sequence. The differences among the Cbeta1 sequences are either allelic polymorphisms or two splice variants, one being a product of exon1 splicing to exon3 (exon2 skipping), and another being an alternative splicing using a splice acceptor site newly discovered inside Cbeta1 exon4. The latter splice acceptor site was also found in human, mouse and horse all giving short cytoplasmic domain with Phe at their C-terminal ends. Other splicing products included trans-splicing of Jbeta2 to Cbeta1, non-functional splicing of two Jbeta gene segments in tandem and a part of Jbeta2.7-Cbeta2 intron to Cbeta2 exon1. Numerous examples of splice variants may suggest the involvement of splicing in generating TCRbeta-chain functional diversity.

Alternative Splicing↗

PubMed2000

Uterine expression of alternatively spliced mRNAs of mouse splicing factor SC35 during early pregnancy.

RNA differential display was applied to identify genes critical for the establishment of pregnancy in the mouse. One of the gene fragments identified was homologous to human SC35 splicing factor; the mouse counterpart had not then been cloned. To obtain the full cDNA sequence of the mouse gene, a cDNA library was screened and four positive clones were fully analysed. Sequencing analysis indicated that we had cloned alternatively spliced mRNA species of mouse SC35 splicing factor. A map of splicing structure for this gene's pre-mRNA was then proposed and region-specific mRNA species were tested on Northern blots. This analysis indicated that the overall expression level of SC35 mRNA was much higher in implantation sites than in inter-implantation sites in the mouse uterus during early pregnancy. The expression of alternatively spliced mRNAs for SC35 was differently regulated both during early pregnancy and by steroid hormones. Embryo-derived factors were also implicated in the up-regulation of SC35 mRNA at implantation sites. These results demonstrate, for the first time, that an essential splicing factor is regulated in a complex manner during implantation in the mouse uterus. Hence, its correct regulation could be important for the success of pregnancy.

Alternative Splicing↗

PubMed2005

Expression of the two alternatively spliced PRKAR1A RNAs in human endocrine glands.

Heterozygous loss of function mutations in human PKAR1A gene (PRKAR1A) have been identified in patients with Carney complex (CNC), an autosomal dominant familial multiple neoplasia syndrome displaying different endocrine tumors, including adrenocortical tumors, GH-secreting pituitary tumors and thyroid adenomas. Although PRKAR1A is encoded by a single gene, it is transcribed from at least two different promoters, adjacent to different first non-coding exons (1a and 1b), giving rise to alternately spliced transcripts coding for identical proteins. The separate regulation of the two distinct promoters and the presence of multiple alternatively spliced first exons suggest a complex mechanism of PRKAR1A expression regulation. In order to investigate the relative expression of the two mRNA transcripts (1a and 1b) in human adult endocrine tissues involved in the determination of CNC phenotype, we selected 17 pituitary, 20 adrenal and seven thyroid tissues from tumoral and peri-tumoral lesion samples. Expression of the two transcripts was evaluated by semi-quantitative RT-PCR and real-time RT-PCR. This study first reports that human pituitary and thyroid tissues show a similar expression of the two transcripts, whereas in adrenal tissues transcript 1b is the most abundant one.

Adrenal Gland Neoplasms↗

PubMed2001

Alternative splicing of Rh blood group polypeptide mRNA produces a novel transcript containing a short nucleotide insertion on human erythroleukemia K562 cells.

A novel isoform of the human Rh blood group polypeptide cDNA was isolated from human erythroleukemic K562 cells. This isoform was produced by deletion of the sequences derived from exons 2 and 3 of the RHCE gene and insertion of 44-bp into the resulting junction between exon 1 and 4 derived sequence. The deduced amino acid sequence revealed that the 44-bp insertion sequence contains an in-frame stop codon that causes premature chain termination. A sequence homology search using GenBank showed that the inserted sequence was derived from the intron between exons 1 and 2 of the RHCE gene. Moreover, analysis of the region surrounding the inserted sequence indicated that the insert was a cryptic exon flanked by consensus donor and acceptor splice sequences. This novel transcript was most likely produced by alternative splicing.

Alternative Splicing↗

PubMed1998

Identification of alternative splicing forms of GLT-1 mRNA in the spinal cord of amyotrophic lateral sclerosis patients.

The glutamate transporter plays an essential role in regulating glutamate levels in the synaptic cleft. It has been postulated that the dysfunction of GLT-1, one subtype of glutamate transporter, may be etiologically related to amyotrophic lateral sclerosis (ALS). Two alternative splicing forms of GLT-1 messenger RNA (mRNA) were found in the cervical spinal cord of five ALS patients and three controls. Analysis with reverse transcription-polymerase chain reaction (RT-PCR) showed that the shorter mRNA was a result of exon 8 skipping. A truncated transcript containing an intronic sequence at the 3' end of exon 7 was also demonstrated. However, the incidence of both alternative mRNAs was not different between the five ALS patients and three controls. Interestingly, the mRNA were also found in the cerebral cortex of a control subject. These results suggest that alternative splicing forms of GLT-1 mRNAs do not play a pathogenetic role in ALS but rather a physiological one in the normal spinal cord and brain.

ATP-Binding Cassette Transporters↗

PubMed1998

Identification, genomic organization, and alternative splicing of KNSL3, a novel human gene encoding a kinesin-like protein.

Proteins of the kinesin superfamily are microtubule-dependent molecular motors that play important roles in organelle transport and cell division. Through genomic sequencing and use of the RT-PCR technique, we have identified and characterized KNSL3 (kinesin-like 3), a novel member of the kinesin-like protein family in humans. We determined its genomic organization and detected four alternatively spliced transcripts. KNSL3 was expressed ubiquitously, but sizes and relative amounts of the major products were different in each of the tissues examined. Alternative splicing, along with the multiplicity of genes in the molecular family that includes KNSL3, produce diversity among the C-terminal ends of kinesins. These observations may contribute to an understanding of the specificity of different kinesins with respect to organelle binding.

Alternative Splicing↗

PubMed1999

Alternative splicing in the murine and human FXR1 genes.

Fragile X syndrome results from mutations in the X-linked FMR1 gene. The most common mutation is expansion and hypermethylation of a CGG repeat in the 5'UTR of FMR1, which blocks transcription and results in the loss of FMR1 protein (FMRP). Efforts to understand the function of FMRP have led to the identification of two autosomal homologs, FXR1P and FXR2P, that may interact with FMRP in some tissues. Reported cDNAs for human, murine, and Xenopus FXR1 suggested the potential for alternatively spliced isoforms, a feature also found in the FMR1 gene. Using RT-PCR to characterize FXR1 alternative splicing in different mouse tissues and human cell lines, we identified seven isoforms that differ by the presence or absence of four DNA regions. These isoforms are found at varying levels in different tissues. The structure of the murine Fxr1h gene underlying these splicing events has also been determined. Interestingly, the longest FXR1P isoform has much greater similarity to FXR2P in the C-terminal region than has been previously recognized, and the gene structure of Fxr1h is quite similar to those of FMR1 and Fxr2h. However, unlike FMR1 and Fxr2h, there is no (CGG)(n) repeat in the 5'UTR region of Fxr1h. Continuing efforts to characterize the expression patterns of FMRP family members should aid in our understanding of their functions in various cells and tissues.

Alternative Splicing↗

PubMed2005

Molecular cloning and expression of novel alternatively spliced cytochrome P450 2E1 mRNAs in humans.

Human cytochrome P450 2E1 (CYP2E1) is a phase I metabolizing enzyme. It is involved in the biotransformation of xenobiotics and endogenous substrates. Inter-individual genetic polymorphisms of the CYP2E1 gene are associated with different cancer diseases as well as alcohol and nicotine dependence. We report here for the first time three novel alternative spliced mRNA transcripts which are more frequently present in lung carcinoma cell lines as in hepatocyte cell lines. They are unexpected detectable in blood leukocytes from healthy volunteers but not in normal and cancerous lung tissue. The full-length wildtype transcript of CYP2E1 is described to be concomitant to an alternatively spliced mRNA transcript. Stimulation with CYP2E1-inducing agents did not change the splicing transcript pattern. The three splicing variants should lead to truncated non-functional proteins. Thus the genetic diversity of CYP2E1 is additionally extended at the transcriptional level of gene expression. The physiological role of the splicing variants is not known, yet, but they seem to be related to the carcinogenic property of the cell lines.

Alleles↗

PubMed2004

Generation of a functional, soluble tapasin protein from an alternatively spliced mRNA.

The loading of newly synthesised MHC class I molecules (MHCI) with peptides requires the involvement of several endoplasmic reticulum (ER)-resident cofactors including calnexin, calreticulin, transporter associated with antigen processing, ERp57 and tapasin. In the absence of tapasin, MHC I complexes are loaded with suboptimal peptides and their recognition by cytotoxic T cells raised to high-affinity, immunodominant peptide epitopes is impaired. Here, we describe the cloning and functional assessment of an alternative spliced form of tapasin. From the EST database, we obtained a partially spliced tapasin cDNA that retained introns 4-6. When transfected into the tapasin-deficient cell line 0.220, the cDNA produced an alternatively spliced tapasin transcript that contained intron 5 (74 bp). This introduced a new stop codon that terminated translation immediately before the putative transmembrane domain and led to a tapasin molecule containing the lumenal domain plus 8 extra novel amino acids at its C-terminus. This molecule promoted peptide loading of HLA-B5 in 0.220 cell line, and restored normal HLA-B5 surface expression. However, the peptides loaded onto HLA-B5 were suboptimal compared to those loaded onto HLA-B5 in the presence of wild-type tapasin.

Alternative Splicing↗

PubMed2005

Co-expression of alternatively spliced forms of PAX3, PAX7, PAX3-FKHR and PAX7-FKHR with distinct DNA binding and transactivation properties in rhabdomyosarcoma.

PAX3 and PAX7 encode transcription factors implicated in the pathogenesis of rhabdomyosarcoma (RMS), including alveolar RMS in which chromosomal translocations generate PAX3-FKHR and PAX7-FKHR fusions. Previous studies of wild-type PAX3 and PAX7 identified alternative splicing events that modify the paired box and generate 2 isoforms of PAX3 (Q+ and Q-) and 4 isoforms of PAX7 (Q+GL+, Q+GL-, Q-GL+, Q-GL-). In our study, we investigated alternative splicing of the wild-type and fusion forms of PAX3 and PAX7 in alveolar and embryonal RMS and assessed the functional implications. For PAX3 and PAX3-FKHR, the Q+ and Q- isoforms were consistently co-expressed in RMS tumors with slightly higher levels of the Q+ isoform. For PAX7 and PAX7-FKHR, there was a consistent pattern of co-expression of the 4 isoforms in RMS tumors: Q+GL- > Q+GL+ >/= Q-GL- > Q-GL+. DNA binding analysis demonstrated that PAX3 and PAX3-FKHR Q- isoforms exhibit higher affinity than corresponding Q+ isoforms for class I sites and no difference for class II sites. For PAX7 and PAX7-FKHR, the relative affinity was Q-GL- > Q+GL- > Q-GL+ >/= Q+GL+ for class I sites and Q-GL-, Q+GL- > Q-GL+, Q+GL+ for class II sites. Finally, the transcriptional activities of the PAX3-FKHR and PAX7-FKHR isoforms on reporter plasmids varied over a 5-fold and 50-fold range, respectively, in accord with the differences in DNA binding activity. In conclusion, these studies reveal that PAX3, PAX7 and their fusions with FKHR are each expressed in RMS tumors as a consistent mixture of functionally distinct isoforms.

Alternative Splicing↗

PubMed2000

Identification of alternative splicing variants of the beta subunit of human Ca(2+)/calmodulin-dependent protein kinase II with different activities.

The beta subunit of human Ca(2+)/calmodulin-dependent protein kinase II (beta CaMKII) was identified by searching through an expressed sequence tag database and rapid amplification of cDNA 5'-ends and was assigned to chromosome 7. Reverse transcription-polymerase chain reaction and sequencing analysis identified at least five alternative splicing variants of beta CaMKII (beta, beta6, betae, beta'e, and beta7) in brain and two of them (beta6 and beta7) were first detected in any species. When expressed in HEK 293 cells, the Ca(2+)/calmodulin-dependent kinase activity of beta7, the shortest variant, was much lower than that of either beta (the longest one) or betae (the medium one), suggesting possible regulation of beta CaMKII activity by alternative splicing.

Alternative Splicing↗

PubMed1995

Functionally different isoforms of the human calcitonin receptor result from alternative splicing of the gene transcript.

Two subtypes of the human calcitonin receptor (hCTR) have been described which differ from one another by the presence or absence of a 16-amino acid insert in the first intracellular loop. Both isoforms were stably expressed in baby hamster kidney cells to compare their ligand binding and second messenger coupling. The binding affinity and the on/off rate of binding for salmon CT were identical for the two receptor isoforms. However, the presence of the insert significantly reduced the ability of the receptor to couple to both adenylate cyclase and phospholipase C. Stimulation of a transient calcium response was only observed with the insert-negative receptor. Similarly, the ED50 for the cAMP response is 100-fold higher for the insert-positive form compared with the insert-negative form of the receptor. However, the maximal cAMP response was equivalent for both receptor isoforms. The rate of internalization of the insert-positive form of the receptor is significantly impaired relative to the insert-negative receptor, which suggests that this process may be dependent on the stimulation of a second messenger pathway. Cloning and characterization of the relevant portion of the hCTR gene revealed that these isoforms are generated by alternative splicing. We also discovered a third isoform of the hCTR, which can be generated by alternative splicing at the same position. The presence of a stop codon in this newly described alternative exon would lead to premature termination of the receptor at the C-terminal end of the first transmembrane domain.

Alternative Splicing↗