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PubMed1995

Molecular cloning, characterization, and genetic mapping of the cDNA coding for a novel secretory protein of mouse. Demonstration of alternative splicing in skin and cartilage.

A novel 85-kDa protein secreted by the mouse stromal osteogenic cell line MN7 was identified using two-dimensional polyacrylamide gel electrophoresis (Mathieu, E., Meheus, L., Raymackers, J., and Merregaert, J. (1994) J. Bone Miner. Res. 9, 903-913). Degenerate primers were used to isolate the cDNA coding for this protein. The full-length cDNA clone is 1.9 kilobases (kb) and codes for a protein of 559 amino acid residues. The DNA and deduced amino acid sequences have no counterparts in public data bases, but a structural similarity involving typical cysteine doublets can be observed to serum albumin family proteins and to Endo16 (a calcium-binding protein of sea urchin). Northern blot analysis revealed the presence of a 1.9-kb transcript in various tissues, and a shorter transcript of 1.5 kb, derived by alternative splicing in tail, front paw and skin of embryonic mice. The gene for the p85 protein, termed Ecm1 (for extracellular matrix protein 1), is a single-copy gene, which was localized to the region on mouse chromosome 3 known to contain at least one locus associated with developmental disorders of the skin, soft coat (soc). Alternative splicing may serve as a mechanism for generating functional diversity in the Ecm1 gene.

Alternative Splicing↗

PubMed2002

Muscle-specific alternative splicing of myotubularin-related 1 gene is impaired in DM1 muscle cells.

The myotubularin-related 1 (MTMR1) gene belongs to a highly conserved family of eucaryotic phosphatases, with at least 11 members in humans. The founder member of this gene family, MTM1, is mutated in X-linked myotubular myopathy, a severe congenital disorder that affects skeletal muscle, and codes for myotubularin, a specific phosphatidylinositol 3-phosphate [PI(3)P] phosphatase. MTM1 and MTMR1 are adjacent on the X chromosome, and the corresponding proteins share 59% sequence identity. In the present study, we investigated the putative role of MTMR1 in myogenesis by analysing its expression pattern in muscle cells during differentiation and in skeletal muscle throughout development. We have identified three novel coding exons in the MTMR1 intron 2 that are conserved between mouse and human, are alternatively spliced, and give rise to six mRNA isoforms. One of the transcripts is muscle-specific and is induced during myogenesis both in vitro and in vivo, and represents the major isoform in adult skeletal muscle. We show that the two main MTMR1 protein muscular isoforms, like myotubularin, efficiently dephosphorylate PI(3)P in vitro. We have also analysed whether MTMR1 alternative splicing is affected in skeletal muscle cells derived from patients with congenital myotonic dystrophy (cDM1), in which mRNA splicing disturbances of specific genes are thought to constitute an important pathogenic mechanism. We found a striking reduction in the level of the muscle-specific isoform and the appearance of an abnormal MTMR1 transcript in differentiated cDM1 muscle cells in culture and in skeletal muscle from cDM1 patients. Our results suggest that MTMR1 plays a role in muscle formation and represents a novel target for abnormal mRNA splicing in myotonic dystrophy.

Alternative Splicing↗

PubMed2002

Identification of hepatopoietin dimerization, its interacting regions and alternative splicing of its transcription.

Hepatopoietin (HPO) is a novel human hepatotrophic growth factor. Recently, we demonstrated that the extracellular ligand form of HPO can stimulate proliferation of hepatic cells via its specific receptor, which is on the surface of these cells. Also, the intracellular form of HPO potentiates the transcriptional factor AP-1. Intriguingly, a variety of HPO complexes with different molecular masses were detected in hepatocytes. In this study, we screened a human fetal liver cDNA library using the yeast two hybrid system with HPO as bait, hoping to find an HPO-binding protein. Surprisingly, HPO, itself, and a previously unidentified alternatively spliced form of HPO (named HPO23) were identified as interacting with HPO, indicating that HPO may exist as a homodimer or heterodimer. These results were further confirmed in vitro and in vivo. First, mass spectrometry analysis demonstrated that recombinant human HPO exist as a homodimer and that disulfide bonds were involved in the formation of rhHPO dimer. Secondly, a pull-down experiment confirmed that GST-HPO and HA-HPO, could bind together in vitro. Using HPO and various HPO deletion mutants, we identified the extreme 30 amino acids at both N- and C-termini are essential for HPO dimerization. Finally, Western blotting analysis demonstrated that HPO exists as two isoforms at 15 kDa and 23 kDa (HPO23) in liver cells, the 15 kDa species is located in nucleus, and the 23 kDa species mainly in the cytoplasm. These results implicated that the capacity of HPO to form both homodimers and heterodimers with the alternatively spliced forms might contribute to the existence of various HPO complexes in hepatic cells.

Alternative Splicing↗

PubMed1994

Structural characterization and alternate splicing of the gene encoding the preadipocyte EGF-like protein pref-1.

Preadipocyte factor 1 (pref-1), a member of the EGF-like protein family, is a transmembrane protein with six tandem EGF-like repeats in the putative extracellular domain. Expression of pref-1 is abolished during the in vitro differentiation of 3T3-L1 preadipocytes to adipocytes, and constitutive expression of pref-1 in preadipocytes inhibits their differentiation [Smas, C.M., & Sul, H.S. (1993) Cell 73, 725-734]. In the present studies, we have isolated and characterized genomic clones for pref-1 and have identified multiple pref-1 transcripts generated by alternate splicing. The pref-1 gene consists of five exons and four introns spanning approximately 7.3 kb. By primer extension analysis, the transcription start site was determined to be 169 bp upstream from the translation initiation codon. We have identified functional promoter sequences by transient transfection using a 2.1 kb fragment of the pref-1 5' flanking region linked to a luciferase gene; the pref-1-luciferase fusion gene construct gave 20-fold higher promoter activity as compared to the promoterless vector. Analysis of exon-intron junctions reveals that unlike the majority of the mammalian EGF-like genes, EGF-like repeats of pref-1 are not encoded by discrete exons. Through RT-PCR and the isolation and analysis of multiple pref-1 cDNA clones, we have identified, in addition to full-length pref-1, five alternately spliced forms with various in-frame deletions of all or a part of the sixth EGF-like repeat, juxta-membrane, and predicted transmembrane domains.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗

PubMed1998

Expression of p210 and p190 BCR-ABL due to alternative splicing in chronic myelogenous leukaemia.

The hallmark of chronic myelogenous leukaemia (CML) is the presence of the Philadelphia chromosome and its resultant fusion message, BCR-ABL, and fusion protein, p210. Patients with CML in blast crisis, or with Philadelphia positive acute lymphoblastic leukaemia (ALL), can have a smaller BCR-ABL fusion transcript possessing only the first exon of BCR fused to ABL. This smaller transcript encodes a 190 kD protein which is more strongly transforming than the p210 protein derived from the larger CML-associated transcript. We performed RT-PCR on samples from CML patients in chronic phase to determine the frequency and mechanism of p190 and p210 co-expression and to see if this correlated with clinical indices. We examined the peripheral blood or marrow of 67 patients with CML and found that 35 of them expressed both transcripts whereas the remainder expressed the p210-encoding transcript exclusively. Additional PCR products of an intermediate size were also frequently detected and have been isolated and sequenced. Data from two of these products indicate that they are the result of alternative splicing and include variable combinations of BCR exons. We believe that the expression of the p190-encoding transcript in the chronic phase of CML is also due to alternative splicing. A comparison of patients co-expressing the p190- and p210-encoding transcripts with those patients who expressed only the p210-encoding transcript detected significantly higher white blood cell (WBC) counts and blast cell counts at time of testing as well as significantly higher white blood cell counts at diagnosis.

Adolescent↗

PubMed2001

Quantitation of alternatively spliced estrogen receptor alpha mRNAs as separate gene populations.

Estrogen receptor (ER) mRNA undergoes alternative splicing generating transcripts that have deletions in various combination of exons. Although several reports have shown that the spliced variant mRNAs are expressed in both normal and malignant tissues, the exact functional role(s) of these molecules have not been established in estrogen induced signal transduction processes mainly due to practical limitations involved in their detection and quantitation. We have recently described a 'Splice Targeted Primer Approach' that can specifically detect splice variants without amplifying the wild type ERs [12]. In the current report, we describe strategies to quantify individual splice variant mRNAs as separate gene populations independent of wild type or other variants using ER exon 7Delta and exon 2Delta as models. We describe the methods of quantifying the exon 7Delta and exon 2Delta transcripts in two breast cancer cell lines, MCF-7 and LCC2, and a breast tumor using the splice-targeted primers in combination with template competition RT PCR. The exon 2Delta splice specific sense primer along with an anti-sense primer in exon 4 amplified a 412 bp product in both cell lines and the tumor that could be quantitated. The exon 7Delta splice targeted anti-sense primer along with a partner primer in exon 2 amplified four transcripts that have deletions in exon 7, exons 7 and 4, exons 7 and 3-4, and exons 7 and 3-5. These four transcripts could be simultaneously quantified by the template competition method described here. Our results also show that the estrogen-independent LCC2 cells express significantly higher levels of the above 7Delta transcripts compared to the estrogen-dependent MCF-7 cells.

Alternative Splicing↗

PubMed1990

CD3 zeta and eta chains are produced by alternative splicing from a common gene.

We have analyzed the genomic organization of the murine CD3 eta gene. As suggested from the sequence of CD3 eta cDNA, exons I-VII are identical to those of CD3 zeta and exon VIII is a unique exon for CD3 eta. Exon VIII of the CD3 eta gene is located 4 kb downstream of exon VIII of the CD3 zeta gene. Together with the existence of a splicing acceptor site just before exon VIII of the CD3 eta gene, the CD3 eta chain is proved to be produced by an alternatively spliced transcript of the common gene of CD3 zeta and eta. Since T cell receptor complex contains two forms of zeta dimers, zeta-zeta and zeta-eta, on a single T cell, which seem to have different functions, the results indicate the importance of analyzing the regulation of the alternative splicing to understand T cell function.

Animals↗

PubMed1998

Modulation of kinase activity and oncogenic properties by alternative splicing reveals a novel regulatory mechanism for B-Raf.

Members of the raf oncogene family encode serine/threonine protein kinases, which activate the mitogen-activated protein kinase kinase MEKs (MAPK or ERK kinases) through direct interaction and phosphorylation. Several recent studies have revealed interesting differences between two members of this family, Raf-1 and B-Raf, regarding their activation, regulation, and kinase activity. In particular, B-Raf was shown to display higher MEK kinase activity than Raf-1. By using both two-hybrid analysis and coimmunoprecipitation experiments, we demonstrate here that B-Raf also markedly differs from Raf-1 by a higher affinity for MEK. We previously reported that the B-raf gene encodes multiple protein isoforms resulting from complex alternative splicing of two exons (exons 8b and 10) located upstream of B-Raf kinase domain. In the present study, we show that these naturally occurring modifications within the protein sequence markedly modulate both the biochemical and oncogenic properties of B-Raf. The presence of exon 10 sequences enhances the affinity for MEK, the basal kinase activity, as well as the mitogenic and transforming properties of full-length B-Raf, whereas the presence of exon 8b sequences seems to have opposite effects. Therefore, alternative splicing represents a novel regulatory mechanism for a protein of the Raf family.

3T3 Cells↗

PubMed1997

Alternative splicing of the human diacylglycerol kinase zeta gene in muscle.

Diacylglycerol can function as a second messenger, and one mechanism for the attenuation of this signal is its conversion to phosphatidic acid, which is catalyzed by diacylglycerol kinase (DGK). We screened a cDNA library from human skeletal muscle and isolated two DGKzeta cDNAs that differed from the 3.5-kb clone originally identified in endothelial cells. One transcript, which was 3.4 kb long, was shown to be nonfunctional; it had a 77-bp deletion that included the translation initiation site. The other was 4.1 kb long with a unique 5' sequence of 853 bp. We also isolated a genomic clone of DGKzeta and determined its organization and location; it contains 32 exons, spans approximately 50 kb of genomic sequence, and maps to chromosome 11p11.2. The protein encoded by the 4.1-kb transcript contains two cysteine-rich regions, a catalytic domain, and ankyrin repeats like the endothelial form of DGKzeta, as well as a unique N-terminal domain. The coding sequence was shown to be derived from alternative splicing of the DGKzeta gene. In cells transfected with the 4.1-kb clone, we detected a 130-kDa protein with an antibody to DGKzeta and demonstrated that it was localized predominantly in the nucleus. We conclude that alternative splicing generates tissue-specific variants of DGKzeta that share some properties but may have unique ones as well.

Alternative Splicing↗

PubMed2006

An alternative splice product of IkappaB kinase (IKKgamma), IKKgamma-delta, differentially mediates cytokine and human T-cell leukemia virus type 1 tax-induced NF-kappaB activation.

NF-kappaB is an inducible transcription factor mediating innate immune responses whose activity is controlled by the multiprotein IkappaB kinase (IKK) "signalsome". The core IKK consists of two catalytic serine kinases, IKKalpha and IKKbeta, and a noncatalytic subunit, IKKgamma. IKKgamma is required for IKK activity by mediating kinase oligomerization and serving to couple the core catalytic subunits to upstream mitogen-activated protein 3-kinase cascades. We have discovered an alternatively spliced IKKgamma mRNA isoform, encoding an in-frame deletion of exon 5, termed IKKgamma-delta. Using a specific reverse transcription-PCR assay, we find that IKKgamma-delta is widely expressed in cultured human cells and normal human tissues. Because IKKgamma-Delta protein is lacking a critical coiled-coil domain important in protein-protein interactions, we sought to determine its signaling properties by examining its ability to self associate, couple to activators of the canonical pathway, and mediate human T-cell leukemia virus type 1 (HTLV-1) Tax-induced NF-kappaB activity. Coimmunoprecipitation and confocal colocalization assays indicate IKKgamma-delta has strong homo- and heterotypic association with wild-type (WT) IKKgamma and, like IKKgamma WT, associates with the IKKbeta kinase. Similarly, IKKgamma-delta mediates IKK kinase activity and downstream NF-kappaB-dependent transcription in response to tumor necrosis factor (TNF) and the NF-kappaB-inducing kinase-IKKalpha signaling pathway. Surprisingly, however, in contrast to IKKgamma WT, IKKgamma-delta is not able to mediate HTLV-1 Tax-induced NF-kappaB-dependent transcription, even though IKKgamma-delta binds and colocalizes with Tax. These observations suggest that IKKgamma-delta is a functionally distinct alternatively spliced mRNA product differentially mediating TNF-induced, but not Tax-induced, signals converging on the IKK signalsome. Differing levels of IKKgamma-delta expression, therefore, may affect signal transduction cascades coupling to IKK.

Alternative Splicing↗

PubMed2003

Alternative splicing of the first intracellular loop of plasma membrane Ca2+-ATPase isoform 2 alters its membrane targeting.

Plasma membrane Ca(2+)-ATPases (PMCAs) are involved in local Ca(2+) signaling and in the spatial control of Ca(2+) extrusion, but how different PMCA isoforms are targeted to specific membrane domains is unknown. In polarized MDCK epithelial cells, a green fluorescent protein-tagged PMCA4b construct was targeted to the basolateral membrane, whereas a green fluorescent protein-tagged PMCA2b construct was localized to both the apical and basolateral domain. The PDZ protein-binding COOH-terminal tail of PMCA2b was not responsible for its apical membrane localization, as a chimeric pump made of an NH(2)-terminal portion from PMCA4 and a COOH-terminal tail from PMCA2b was targeted to the basolateral domain. Deletion of the last six residues of the COOH terminus of either PMCA2b or PMCA4b did not alter their membrane targeting, suggesting that PDZ protein interactions are not essential for proper membrane localization of the pumps. Instead, we found that alternative splicing affecting the first cytosolic loop determined apical membrane targeting of PMCA2. Only the "w" form, which contains a 45-amino acid residue insertion, showed prominent apical membrane localization. By contrast, the x and z splice variants containing insertions of 14 and 0 residues, respectively, localized to the basolateral membrane. The w splice insert was the crucial determinant of apical PMCA2 localization, and this was independent of the splice configuration at the COOH-terminal end of the pump; both PMCA2w/b and PMCA2w/a showed prominent apical targeting, whereas PMCA2x/b, PMCA2z/b, and PMCA2z/a were confined to the basolateral membrane. These data report the first differential effect of alternative splicing within the first cytosolic loop of PMCA2 and help explain the selective enrichment of specific PMCA2 isoforms in specialized membrane compartments such as stereocilia of auditory hair cells.

Alternative Splicing↗

PubMed2001

JC virus T' proteins encoded by alternatively spliced early mRNAs enhance T antigen-mediated viral DNA replication in human cells.

Alternative splicing of the JC Virus (JCV) precursor early mRNA yields five transcripts that encode proteins that regulate the life cycle of this human polyomavirus. Large T protein (TAg) mediates viral DNA replication and oncogenic activities, and small t protein influences these functions under certain conditions. Recently, three new early proteins, T'(135), T'(136), and T'(165), were discovered that contain sequences overlapping amino-terminal TAg functional domains. Initial studies with the T' proteins suggested they contribute to viral DNA replication and transformation. Mutation of a donor splice site utilized by all three T' mRNAs creates a mutant that exhibits a 10-fold decrease in viral DNA replication compared to wild type JCV. To assess the influence that individual T' proteins have on the replication process, a set of T' acceptor site mutants was created in which the unique second acceptor splice site of each T' mRNA was altered to eliminate production of one, two or all three T' mRNAs. The patterns of early mRNA and protein expression in these seven mutants were examined, and it was found that mutation of the T'(135) acceptor site resulted in the utilization of cryptic splice sites and the generation of new T' species. Additional mutations were made to prevent these aberrant splicing reactions prior to measuring DNA replication potential of the mutants. DpnI assays revealed that each T' protein contributes to TAg-mediated DNA replication activity. The three single mutants that express two T' proteins and the double mutant that only produces T'(136), exhibited levels of replication equivalent to that of wild type virus, whereas the two double mutants that fail to express T'(136) replicated about twofold less efficiently than wild-type JCV. Replication activity of the triple acceptor site mutant, like that of the T' donor site mutant from an earlier study, was impaired significantly.

Alternative Splicing↗

PubMed2000

The nonsense-mediated decay pathway and mutually exclusive expression of alternatively spliced FGFR2IIIb and -IIIc mRNAs.

Exons IIIb and IIIc of the FGFR2 gene are alternatively spliced in a mutually exclusive manner in different cell types. A switch from expression of FGFR2IIIb to FGFR2IIIc accompanies the transition of nonmalignant rat prostate tumor epithelial cells (DTE) to cells comprising malignant AT3 tumors. Here we used transfection of minigenes with and without alterations in reading frame and with and without introns to examine how translation affects observed FGFR2 splice products. We observed that nonsense mutations in other than the last exon led to a dramatic reduction in mRNA that is abrogated by removal of downstream introns in both DTE and AT3 cells. The mRNA, devoid of both IIIb and IIIc exons (C1-C2), is a major splice product from minigenes lacking an intron downstream of the second common exon C2. From these observations, we suggest that repression of exon IIIc and activation of exon IIIb inclusion in DTE cells lead to the generation of both C1-IIIb-C2 and C1-C2 products. However, the C1-C2 product from the native gene is degraded due to a frameshift and a premature termination codon caused by splicing C1 and C2 together. Derepression of exon IIIc and repression of exon IIIb lead to the generation of both C1-IIIc-C2 and C1-C2 products in AT3 cells, but the C1-C2 product is degraded. The C1-IIIb-IIIc-C2 mRNA containing a premature termination codon in exon IIIc was present, but at apparently trace levels in both cell types. The nonsense-mediated mRNA decay pathway and cell type-dependent rates of inclusion of exons IIIb and IIIc result in the mutually exclusive expression of FGFR2IIIb and IIIc.

Alternative Splicing↗

PubMed2002

Structural analysis of the DNA-binding domain of alternatively spliced steroid receptors.

We have generated 24 DNA-binding domain structure models of alternatively spliced or mutated steroid receptor variants by homology-based modeling. Members of the steroid receptor family dispose of a DNA-binding domain which is built from two zinc fingers with a preserved sequence homology of about 90%. Data from crystallographic analysis of the glucocorticoid receptor DNA-binding domain are therefore appropriate to serve as a template structure. We inserted or deleted amino acid residues in order to study the structural details of the glucocorticoid, mineralocorticoid, and androgen receptor splice variants. The receptor variants are created by QUANTA- and MODELLER-based modeling. Subsequently, the resulting energy-minimized models were compared with each other and with the wild-type receptor respectively. A prediction for the receptor function based mainly on the preservation or destruction of secondary structures has been carried out. The simulations showed that amino acid insertions of one, four or nine additional residues of existing steroid receptor splice variants were tolerated without destruction of the secondary structure. In contrast, a deletion of four amino acids at the splice site junction leads to modifications in the secondary structure of the DNA-recognition helix which apparently disturb the receptor-DNA interaction. Furthermore, an insertion of 23 amino acid residues between the zinc finger of the androgen receptor leads to a large loop with an additional alpha-helical structure which seems to disconnect a specific contact from its hormone response element. Thereafter, the prediction of receptor function based on the molecular models was compared with the available experimental results from the in vitro function tests. We obtained a close correspondence between the molecular modeling-based predictions and the conclusions of receptor function drawn from in vitro studies.

Alternative Splicing↗

PubMed1997

Mutations and alternative splicing of the BRCA1 gene in UK breast/ovarian cancer families.

BRCA1 is a tumour suppressor gene located on chromosome band 17q21. It is estimated that mutations in the BRCA1 gene account for approximately 45% of the breast cancer families and almost all of the breast/ovarian cancer families. We have used single strand conformation polymorphism analysis, direct sequencing, allele specific oligonucleotide hybridisation, and reverse transcription polymerase chain reaction (RT-PCR) to look for mutations in the BRCA1 gene in 49 breast or breast/ovarian cancer families. Five distinct mutations, three novel and two previously observed, were detected in seven families. Each novel mutation was identified in one family: 3896delT in exon 11, a splicing mutation in the intron 9-exon 10 junction, and an inferred regulatory mutation. The 185delAG in exon 2 was found in three families sharing the same haplotype, but this haplotype is different from that shared by the Ashkenazi Jewish families, suggesting that the 185delAG in our families may have arisen independently. Another previously reported mutation, the 3875del4 in exon 11, was identified in one family. Of the 49 families examined, linkage analyses for both the BRCA1 and the BRCA2 regions were performed on 33 families, and mutations in the BRCA1 gene were identified in all but one family that have a lod score above 0.8 for BRCA1. All of the mutations cause either a truncated BRCA1, or loss of a BRCA1 transcript, thus are likely to be functionally disruptive. In addition, we found that alternative splicing is a common phenomenon in the processing of the BRCA1 gene. Seven variant BRCA1 transcripts were identified by RT-PCR; all but one maintained the BRCA1 open reading frame. We believe that alternative splicing may play a significant role in modulating the physiological function of BRCA1.

Alternative Splicing↗

PubMed2000

Regulation of telomerase by alternate splicing of human telomerase reverse transcriptase (hTERT) in normal and neoplastic ovary, endometrium and myometrium.

Telomerase extends telomeric repeats at the ends of linear chromosomes, thereby prolonging the replicative capacity of cells. To investigate possible regulatory mechanisms of telomerase, we measured telomerase enzyme activity, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA in normal and neoplastic ovary, endometrium and myometrium. Telomerase activity was detected in most malignancies and in normal endometrium but not in myometrial leiomyoma, normal myometrium or normal ovary. hTR was expressed in all tissue samples. hTERT mRNA was expressed in many tissue samples, and no tissue sample exhibited telomerase activity without expressing hTERT mRNA. However, the presence of hTR and hTERT mRNA was not sufficient for telomerase activity. Alternate splicing of hTERT produced mRNAs lacking critical reverse transcriptase (RT) motifs in both normal and neoplastic tissues. Only tissues expressing hTERT containing complete A and B RT motifs demonstrated telomerase activity. Finally, several normal ovarian tissues and myometrial leiomyomas lacked telomerase activity despite expressing hTR and hTERT containing complete A and B RT motifs. This was not seen in ovarian and myometrial malignancies, where the expression of hTR and hTERT containing complete A and B RT motifs was sufficient for telomerase activity. We conclude that in ovarian and uterine tissues, the presence of a functional telomerase complex is regulated at multiple levels, including hTERT transcription and alternative splicing of hTERT transcripts. The lack of telomerase activity in several normal but not malignant tissues expressing hTR and hTERT containing complete A and B RT motifs suggests that there are further mechanisms for suppressing telomerase activity downstream of hTERT transcription and mRNA splicing, and these mechanisms have been lost during neoplastic transformation.

Adenocarcinoma↗

PubMed1994

Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase: genomic organization, alternative splicing, and structure/function predictions.

Wilson disease is an autosomal recessive disorder of copper transport. Disease symptoms develop from the toxic build-up of copper primarily in the liver, and subsequently in the brain, kidney, cornea and other tissues. A candidate gene for WD (ATP7B) has recently been identified based upon apparent disease-specific mutations and a striking amino acid homology to the gene (ATP7A) responsible for another human copper transport disorder, X-linked Menkes disease (MNK). The cloning of WD and MNK genes provides the first opportunity to study copper homeostasis in humans. A preliminary analysis of the WD gene is presented which includes: isolation and characterization of the 5'-end of the gene; construction of a genomic restriction map; identification of all 21 exon/intron boundaries; characterization of extensive alternative splicing in brain; prediction of structure/function features of the WD and MNK proteins which are unique to the subset of heavy metal-transporting P-type ATPases; and comparative analysis of the six metal-binding domains. The analysis indicates that WD and MNK proteins belong to a subset of transporting ATPases with several unique features presumably reflecting their specific regulation and function. It appears that the mechanism of alternative splicing serves to regulate the amount of functional WD protein produced in brain, kidney, placenta, and possibly in liver.

Adenosine Triphosphatases↗

PubMed2004

Alternative splicing mechanisms for the modulation of protein function: conservation between human and other species.

Alternative splicing (AS) is an important process in eukaryotic organisms by which a given gene may express a set of different protein isoforms depending on the tissue, or the developmental stage of the individual. In the present work, we have compared AS among species, focusing on the conservation of AS mechanisms for the modulation of protein function. For this purpose, we first analysed the frequency with which different species, human, mouse, rat and fruitfly, utilise them. Second, we focused more directly on the conservation among species of the mechanisms themselves. To this end, we compared biologically equivalent AS events between human and mouse, or rat. Our results indicate only minor differences in the frequency of use of these mechanisms, as well as a high degree of conservation among the species studied.

Alternative Splicing↗