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PubMed1998

Detection of mRNAs and alternatively spliced transcripts of dopamine receptors in rat peripheral sensory and sympathetic ganglia.

The presence of mRNAs for dopamine receptor subtypes and dopamine transporter in rat peripheral sensory and sympathetic ganglia was investigated using polymerase chain reaction (PCR) and DNA sequencing. Dopamine D1, D2, D3, D5 receptor subtype mRNAs and dopamine transporter mRNA were detected in both superior cervical sympathetic ganglia (SCG) and dorsal root ganglia (DRG) in the rat; the expression of D4 mRNA was only detected in DRG. While two alternatively spliced isoforms of D2 were detected in both ganglia, the alternative splicing transcripts for D3 and D4 were only found in the DRG. These results are useful in further studying the roles of dopamine and the effects of dopaminergic agents in the peripheral nervous system.

Alternative Splicing↗

PubMed1998

Functional characterization of alternatively spliced human SERCA3 transcripts.

The sarcoplasmic (or endoplasmic) reticulum Ca2+-ATPase (SERCA)-3 has been implicated in the possible dysregulation of Ca2+ homeostasis that accompanies the pathology of hypertension and diabetes. We report the molecular cloning of two alternatively spliced transcripts from the human SERCA3 gene, ATP2A3, that encode proteins that differ at their carboxy termini by 36 amino acids. SERCA3 transcripts were most abundantly expressed in lymphoid tissues, intestine, pancreas, and prostate. The two human SERCA3 proteins encoded by alternatively spliced transcripts were recognized by the monoclonal antibody PL/IM430 and demonstrated Ca2+ uptake and ATPase activity with an apparent Ca2+ affinity 0.5 pCa unit lower than that of other SERCA gene products. The subcellular distribution of SERCA3 protein was indistinguishable from that of SERCA2b, with expression in the nuclear envelope and in the endoplasmic reticulum throughout the cell. Two variant SERCA3 constructs, huS3-I and huS3-II, were isolated that encode proteins with three amino acid differences: Ala-673 (in huS3-I) substituted for Thr (in huS3-II), Ile-817 substituted for Met, and an insertion of Glu-994. huS3-I displayed a 10-fold lower capacity to transport Ca2+ than huS3-II.

Alternative Splicing↗

PubMed1995

Temporally distinctive changes of alternative splicing patterns during myogenic differentiation of C2C12 cells.

It is well known that skeletal muscle differentiation is accompanied by the appearance of many muscle-specific components and that some of these components are generated through muscle-specific alternative splicing. It is not clear, however, in what manner, including timing, the system that regulates the muscle-specific splicing reactions is constructed during the process of myogenic differentiation. We simultaneously examined the changes in several splicing patterns for the neural cell adhesion molecule (NCAM), beta-tropomyosin, and M-type pyruvate kinase genes during myogenic differentiation of cultured myoblasts using the reverse transcription-polymerase chain reaction method. The NCAM glycosylphosphatidylinositol anchor form increased in preference to the transmembrane form immediately after the induction of differentiation, while the selection of NCAM MSD1 (muscle-specific domain 1) exons started and abruptly increased at about the time when cell-fusion appeared. M2-type pyruvate kinase was gradually substituted for the M1-type molecule. Skeletal muscle-type beta-tropomyosin was predominantly selected even in myoblasts in the growth medium. As a result, each transcript of these genes independently showed a temporally distinctive pattern of change in isoform selecting during the myogenic differentiation of C2C12 cells. These observations suggest that some independent regulation of alternative splicing reactions should occur during myogenic differentiation.

Alternative Splicing↗

PubMed1997

The gene for the human interleukin-11 receptor alpha chain locus is highly homologous to the murine gene and contains alternatively spliced first exons.

The gene for the murine interleukin-11 receptor alpha chain (mIL-11R alpha) contains two loci (1 and 2), of which locus 2 is restricted to only some mouse strains. Two alternatively spliced exons (1a and 1b) encode the 5' untranslated region (5'UTR) of the murine locus 1. We have characterized the gene for the human interleukin-11 receptor alpha chain locus (hIL-11R alpha), examined its expression by Northern analysis and determined its chromosomal location by fluorescence in situ hybridization. The presence of exon(s) encoding the 5'UTR and mapping of transcription initiation sites was determined by reverse-transcriptase polymerase chain reaction and 5' rapid amplification of cDNA ends (5'RACE) techniques. The human locus spanned 10 kilobasepairs (kb) and consisted of 14 exons. Two alternatively spliced first exons (1a and 1b) encoding the 5'UTR were identified and shared 76 and 73% nucleotide identity with murine exons 1a and 1b. Multiple transcription start sites were demonstrated for human exon 1a. The promoter regions of both human exons 1a and 1b did not display a canonical TATA box. A predominant 1.8 kb transcript for the hIL-11R alpha was present in heart, brain, skeletal muscle, lymph nodes, thymus, appendix, pancreas and foetal liver. The hIL-11R alpha gene was localized to chromosome 9p13. In summary, the hIL-11R alpha gene was highly related to locus 1 of the murine gene and there was no evidence of a second hIL-11R alpha locus.

Alternative Splicing↗

PubMed1995

Antisense mapping the MOR-1 opioid receptor: evidence for alternative splicing and a novel morphine-6 beta-glucuronide receptor.

Although MOR-1 encodes a mu opioid receptor, its relationship to the pharmacologically defined mu receptor subtypes has been unclear. Antisense mapping now suggests that these subtypes result from alternative splicing of MOR-1. Three oligodeoxynucleotide probes targeting exon 1 and another oligodeoxynucleotide directed against the coding region of exon 4 block supraspinal morphine analgesia, a mu1 action, while five of six oligodeoxynucleotides directed against exons 2 and 3 are inactive. Inhibition of gastrointestinal transit and spinal morphine analgesia, two mu2 actions, are blocked only by the probe against exon 4 and not by those directed against exon 1. In contrast, the analgesic actions of the extraordinarily potent mu drug morphine-6 beta-glucuronide are blocked by six different antisense oligodeoxynucleotides targeting exons 2 and 3, but not by those acting on exons 1 or 4. These results suggest that the mu1 and mu2 receptor subtypes originally defined in binding and pharmacological studies result from alternative splicing of MOR-1 while morphine-6 beta-glucuronide acts through a novel, previously unidentified receptor which is yet another MOR-1 splice variant.

Alternative Splicing↗

PubMed1999

The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.

G protein-coupled receptor kinases (GRKs) desensitize G protein-coupled receptors by phosphorylating activated receptors. The six known GRKs have been classified into three subfamilies based on sequence and functional similarities. Examination of the mouse GRK4 subfamily (GRKs 4, 5, and 6) suggests that mouse GRK4 is not alternatively spliced in a manner analogous to human or rat GRK4, whereas GRK6 undergoes extensive alternative splicing to generate three variants with distinct carboxyl termini. Characterization of the mouse GRK 5 and 6 genes reveals that all members of the GRK4 subfamily share an identical gene structure, in which 15 introns interrupt the coding sequence at equivalent positions in all three genes. Surprisingly, none of the three GRK subgroups (GRK1, GRK2/3, and GRK4/5/6) shares even a single intron in common, indicating that these three subfamilies are distinct gene lineages that have been maintained since their divergence over 1 billion years ago. Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates over the past 100 million years. The divergence of individual GRKs at vastly different rates reveals that strikingly different evolutionary pressures apply to the function of the individual GRKs.

Alternative Splicing↗

PubMed1996

Structure and alternative splicing of the presenilin-2 gene.

Missense mutations in the presenilin-1 (PS-1) and presenilin-2 (PS-2) genes have been shown to be causes of autosomal dominant Alzheimer's disease (the AD3 and AD4 loci, respectively). Alternative splicing has previously been reported in the PS-1 gene. In this study, elucidation of intron/exon boundary sequences revealed that PS-2 is encoded by 10 coding exons. In addition, PS-2 cDNA cloning and RT-PCR using RNA from a variety of normal tissues revealed the presence of alternatively spliced products. These products included species with in frame omissions of exon 8 and simultaneous omissions of exons 3 and 4.

Alternative Splicing↗

PubMed1994

Tissue-specific alternative splicing of neurofibromatosis 1 (NF1) mRNA.

The neurofibromatosis 1 gene NF1 appears to play a crucial role in regulating the proliferation of cells of neural crest origin. The NF1 gene is a 300 kbp gene, encoding a complex pattern of mRNA related to the presence or absence of two alternative splices. The first splice, in the centre of the coding region of the gene, results in the addition of 63 bp in the GAP-related domain. The second splice located 4203 bp downstream, near the 3' terminus of the coding region of the gene, consists of a 54 bp insert. RT-PCR analysis demonstrates that the most prevalent splice variant in human tissues is the one which contains the GAP-related splice and omits the 3' terminal splice. It is also the form expressed in the peripheral nerve, adrenal medulla, benign NF1 neurofibromas and NF1 neurosarcomas. Conversely, a few organs (brain, muscle) exhibit extensive alternative splicing leading to the co-expression of four distinct transcripts. The reproducibility of the relative levels of each of the splice types in the different organs indicates a tissue-specific splicing pattern of the NF1 gene.

Alternative Splicing↗

PubMed2004

A frameshift mutation and alternate splicing in human brain generate a functional form of the pseudogene cytochrome P4502D7 that demethylates codeine to morphine.

A frameshift mutation 138delT generates an open reading frame in the pseudogene, cytochrome P4502D7 (CYP2D7), and an alternate spliced functional transcript of CYP2D7 containing partial inclusion of intron 6 was identified in human brain but not in liver or kidney from the same individual. mRNA and protein of the brain variant CYP2D7 were detected in 6 of 12 human autopsy brains. Genotyping revealed the presence of the frameshift mutation 138delT only in those human subjects who expressed the brain variant CYP2D7. Genomic DNA analysis in normal volunteers revealed the presence of functional CYP2D7 in 4 of 8 individuals. In liver, the major organ involved in drug metabolism, a minor metabolic pathway mediated by CYP2D6 metabolizes codeine (pro-drug) to morphine (active drug), whereas norcodeine is the major metabolite. In contrast, when expressed in Neuro2a cells, brain variant CYP2D7 metabolized codeine to morphine with greater efficiency compared with the corresponding activity in cells expressing CYP2D6. Morphine binds to micro-opioid receptors in certain regions of the central nervous system, such as periaqueductal gray, and produces pain relief. The brain variant CYP2D7 and micro-opioid receptor colocalize in neurons of the periaqueductal gray area in human brain, indicating that metabolism of codeine to morphine could occur at the site of opioid action. Histio-specific isoforms of P450 generated by alternate splicing, which mediate selective metabolism of pro-drugs within tissues, particularly the brain, to generate active drugs may play an important role in drug action and provide newer insights into the genetics of metabolism.

Alternative Splicing↗

PubMed1988

Multiple transcripts of the mouse tyrosinase gene are generated by alternative splicing.

We report the cloning and isolation of the mouse tyrosinase cDNA by screening mouse B16 melanoma cDNA libraries. Independent cDNA clones have been characterized by restriction enzyme analysis, hybridizations with individual subprobes and by partial sequencing analysis. Based on these criteria we have identified multiple transcripts, which in comparison to the major transcript, display deletions of internal sequences and have different 3' termini. The most abundant transcript encodes a functional tyrosinase. The structural gene which encodes five exons separated by large introns and spans a chromosomal region of approximately 70 kb has been isolated. Comparison of the cDNAs with the cloned genomic DNAs and sequencing of the exon/intron boundaries reveal that the multiple transcripts are generated by alternative splicing and putatively by alternate polyadenylation site usage. The alternative splicing mechanisms involve exon skipping as well as internal donor splice site usage. Primer extension analysis shows that the transcripts are produced from two different promoters. Southern blot analysis of DNAs derived from mice carrying the lethal albino deletion mutations demonstrates that the structural gene maps near or at the albino locus. The viable albino mouse BALB/c carries an apparently intact structural gene indicating that the albino phenotype is a consequence of a failure to express the tyrosinase gene or the inability to produce a tyrosinase enzyme.

Animals↗

PubMed1995

The human aromatic L-amino acid decarboxylase gene can be alternatively spliced to generate unique protein isoforms.

Aromatic L-amino acid decarboxylase (AADC) is expressed in a wide variety of tissues, including those where it is known to convert L-DOPA and 5-hydroxytryptophan to dopamine and serotonin, respectively. AADC has been cloned from many species and shown to undergo alternative splicing within its 5' untranslated region. Here, we report that the human AADC gene can undergo additional alternative splicing of exon 3, generating two different protein isoforms (termed AADC480 and AADC442). Both transcripts are widely expressed, with AADC442 predominating in many neuronal and nonneuronal tissues. When homogenates were prepared from COS-7 cells transfected with expression vectors containing either cDNA, AADC480 catalyzed the decarboxylation of both L-DOPA and 5-hydroxytryptophan. AADC442 was inactive in either assay. These findings suggested that AADC442 may have a different function in non-monoamine-expressing tissues. Taken together, these results suggest that the human AADC gene undergoes complex processing, leading to the formation of both tissue-specific transcripts as well as unique protein isoforms.

5-Hydroxytryptophan↗

PubMed2000

Alternative splicing of the human Rab6A gene generates two close but functionally different isoforms.

Analysis of the human Rab6A gene structure reveals the presence of a duplicated exon, and incorporation of either of the two exons by alternative splicing is shown to generate two Rab6 isoforms named Rab6A and Rab6A', which differ in only three amino acid residues located in regions flanking the PM3 GTP-binding domain of the proteins. These isoforms are ubiquitously expressed at similar levels, exhibit the same GTP-binding properties, and are localized to the Golgi apparatus. Overexpression of the GTP-bound mutants of Rab6A (Rab6A Q72L) or Rab6A' (Rab6A' Q72L) inhibits secretion in HeLa cells, but overexpression of Rab6A' Q72L does not induce the redistribution of Golgi proteins into the endoplasmic reticulum. This suggests that Rab6A' is not able to stimulate Golgi-to-endoplasmic reticulum retrograde transport, as described previously for Rab6A. In addition, Rab6A' interacts with two Rab6A partners, GAPCenA and "clone 1," but not with the kinesin-like protein Rabkinesin-6, a Golgi-associated Rab6A effector. Interestingly, we found that the functional differences between Rab6A and Rab6A' are contingent on one amino acid (T or A at position 87). Therefore, limited amino acid substitutions within a Rab protein introduced by alternative splicing could represent a mechanism to generate functionally different isoforms that interact with distinct sets of effectors.

Alternative Splicing↗

PubMed2005

Nonsense-associated alternative splicing of the human thyroglobulin gene.

INTRODUCTION: We have described in previous articles a nonsense mutation (4588C>T, R1511X) in exon 22 of the thyroglobulin (TG) gene in a member of a family with a complex history of congenital goiter. In the mutated thyroid gland, full-length thyroglobulin mRNA is almost undetectable. However, a smaller transcript is detected in which the mutated exon 22 is skipped and the reading frame restored. It is conceivable that alternative splicing might be a mechanism involved in the rescue of nonsense mutations. METHODS: To investigate whether the detection of the alternative mRNA is due to an increase in its concentration or its preferential amplification during reverse transcriptase-PCR in the absence of the normal full-length mRNA competitor, we set up an assay in which the competitor mRNA was provided. We also studied the effect of the 4588C>T mutation on exon definition and processing using wild-type and mutated minigenes. RESULTS: The detection of the alternative mRNA lacking exon 22 is not caused by the absence of the full-length competitor. In contrast, our results demonstrate that the alternative transcript preferentially accumulates in the mutated thyroid at a level similar to the full-length transcript in control tissue. Transient expression experiments with wild-type and mutated minigenes indicate that the mutated exon is as efficiently spliced as the wild-type, suggesting that the 4588C>T mutation does not interfere with exon 22 definition and processing. CONCLUSIONS: The alternative splicing of the TG gene described in this article constitutes a new case of nonsense-associated alternative splicing. We have shown that the mutation itself does not interfere with exon definition and processing in vitro. Our results support the hypothesis that the alternative splicing of the mutated exon is driven by the interruption of the reading frame.

Alternative Splicing↗

PubMed1988

Both VP2 and VP3 are synthesized from each of the alternative spliced late 19S RNA species of simian virus 40.

The late 19S RNAs of simian virus 40 consist of a family of alternatively spliced RNAs, each of which contains open reading frames corresponding to all three of the virion proteins. Two approaches were used to test the hypothesis that each alternatively spliced 19S RNA species is translated to synthesize preferentially only one of the virion proteins. First, we analyzed the synthesis of virion proteins in simian virus 40 mutant-infected monkey cells that accumulate predominantly either only one spliced 19S RNA species or only the 19S RNAs. Second, we determined the virion proteins synthesized in a rabbit reticulocyte lysate programmed with specific, in vitro-transcribed 19S RNA species. These results indicated that VP2 and VP3, but not VP1, are synthesized from all 19S RNA species. Quantitative analysis of these data indicated that individual 19S RNA species containing a translation initiation signal upstream of the VP2 AUG codon were translated in a cell extract three- to fivefold less efficiently than were 19S RNA species lacking this signal and that the precise rate of synthesis of VP2 relative to VP3 varied somewhat with the sequence of the leader region. These data are consistent with the synthesis of VP2 and VP3 occurring by a leaky scanning mechanism in which initiation of translation at a specific AUG codon is affected by both (i) the intrinsic efficiency of ribosomes recognizing the sequences surrounding the AUG codon as an initiation signal and (ii) partial interference from 5'-proximal initiation signals and their corresponding open reading frames.

Animals↗

PubMed1995

Augmented DNA-binding activity of p53 protein encoded by a carboxyl-terminal alternatively spliced mRNA is blocked by p53 protein encoded by the regularly spliced form.

DNA-binding activity of the wild-type p53 is central to its function in vivo. However, recombinant or in vitro translated wild-type p53 proteins, unless modified, are poor DNA binders. The fact that the in vitro produced protein gains DNA-binding activity upon modification at the C terminus raises the possibility that similar mechanisms may exist in the cell. Data presented here show that a C-terminal alternatively spliced wild-type p53 (ASp53) mRNA expressed by bacteria or transcribed in vitro codes for a p53 protein that efficiently binds DNA. Our results support the conclusion that the augmented DNA binding activity of an ASp53 protein is probably due to attenuation of the negative effect residing at the C terminus of the wild-type p53 protein encoded by the regularly spliced mRNA (RSp53) rather than acquisition of additional functionality by the alternatively spliced C' terminus. In addition, we found that ASp53 forms a complex with the non-DNA-binding RSp53, which in turn blocks the DNA-binding activity of ASp53. Interaction between these two wild-type p53 proteins may underline a mechanism that controls the activity of the wild-type p53 protein in the cell.

Alternative Splicing↗

PubMed1996

Myelin/oligodendrocyte glycoprotein is alternatively spliced in humans but not mice.

Myelin/oligodendrocyte glycoprotein (MOG) is an integral membrane protein expressed on the oligodendrocyte cell surface and the outermost surface of myelin sheaths. Due to this localization, MOG is a primary target antigen involved in immune-mediated demyelination. We previously reported that MOG is a unique member of the immunoglobulin (Ig) superfamily in that it possesses two large hydrophobic domains. MOG is highly conserved between deduced peptide sequences of rodent and human MOG (approximately 89% identity). We have completed an investigation of alternative splicing within the human and mouse MOG genes. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of total cellular RNA isolated from both fetal and adult human central nervous system (CNS) tissues reveals a complex array of alternatively spliced MOG-specific variants and the presence of two novel exons. Exon 3 encodes a short hydrophilic domain containing multiple in-frame termination codons that would result in truncation of MOG prior to translation of its transmembrane domain. Exon 7 encodes an additional hydrophilic domain that replaces MOG's second hydrophobic domain in one splice variant. We also observed that five of our eight MOG variants exhibited an alternative internal 3' splice acceptor within MOG's terminal exon. Surprisingly, no splicing was observed in a developmental study using mouse brainstem RNA.

Alternative Splicing↗

PubMed1993

Expression and alternative splicing of fibronectin mRNA in human diploid endothelial cells during aging in vitro.

Different mRNAs for fibronectin arise from the variable processing of a single primary transcript. We used ribonuclease protection assay to investigate the changes occurring in fibronectin expression and the alternative splicing of mRNA precursor during aging in vitro of human diploid endothelial cells. Senescent endothelial cells release more protein and contain 4-5-fold more fibronectin mRNA than young cells. The pattern of alternative splicing of fibronectin mRNA, with the EDA and the CS1 segments largely included (35% and 77%, respectively) and the EDB segment undetectable, correlates well with previous studies at the protein level both in vitro and in vivo. No changes in the splicing pattern of fibronectin mRNA precursor were detected during endothelial cellular senescence. The increased expression of fibronectin in senescent cells may be a result of the activity of interleukin-1 alpha, which is overexpressed in senescent endothelial cells. It could be also important in vivo during aging and in atherosclerotic lesions.

Alternative Splicing↗

PubMed1992

Alternative splicing in the human lck gene leads to the deletion of exon 1' and results in a new type II lck transcript.

In this study we describe a new lck mRNA transcribed from the type II promoter, resulting from alternative splicing, in the deletion of exon 1' which comprises the AUG initiation codon. In this type IIB lck transcript, as a consequence of exon 1' deletion, a new AUG codon in exon 1, located 35 nt upstream from the regular initiation codon in exon 1', and normally out of frame, is now in frame with exon 2 and the following lck coding sequence. In this type IIB lck transcript, 10 residues encoded by exon 1 from the new AUG codon replace the first 35 residues encoded by exon 1'. Strikingly, in this new N-terminal amino acid sequence, a glycine residue, needed for myristylation and anchorage in the plasma membrane, is present at position 2. This alternative splicing has been observed in T-cell lines, normal mature T cells and in peripheral blood lymphocytes from different leukemic patients. Different ratios of regular type IIA lck mRNA (with exon 1') to spliced type IIB lck mRNA (without exon 1') have been observed depending on the patient.

Alternative Splicing↗