Evidence For Alternative Splicing Research Articles

Antisense mapping the MOR-1 opioid receptor: evidence for alternative splicing and a novel morphine-6β-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 results 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 mu 1 action, while five of six oligodeoxynucleotides directed against exons 2 and 3 are inactive. Inhibition of gastrointestinal transit and spinal morphine analgesia, two mu 2 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-6β-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 mu 1 and mu 2 receptor subtypes originally defined in binding and pharmacological studies result from alternative splicing of MOR-1 while morphine-6β-glucuronide acts through a novel, previously unidentified receptor which is yet another MOR-1 splice variant.

Friedreich's ataxia: a defect in signal transduction?

We have previously assigned the mutation causing Friedreich's ataxia (FRDA) to 9q13 by genetic linkage and fluorescent in situ hybridization analysis, and identified recombination events which position the gene centromeric to D9S5. We report here the extension of a yeast artificial chromosome contig to span the 860 kb interval immediately proximal to this marker, which includes the D9S886 and D9S887/888 loci reported to flank the FRDA locus, and the construction of a high resolution cosmid contig initiated from the D9S888 locus. Exon trapping and cDNA library screening strategies have resulted in the isolation of a candidate gene which traverses the centromeric boundary of the FRDA critical region. The gene spans a genomic interval greater than 220 kb with at least two of the coding exons located proximal to the D9S887/888 loci. Expression is complex, with multiple transcripts detected in a variety of tissues and evidence of alternative splicing and developmental control. The predicted amino acid sequence for the 2.7 kb transcript reported here shows a marked homology to the deduced amino acid sequence of the Saccharomyces cerevisiae MSS4 protein, proposed to function within the phosphoinositide cycle, suggesting a potential role for the human homologue in signal transduction. Whilst no evidence for mutation has been detected in this transcript, the sequence represents only one of the shorter alternatively spliced species identified by Northern analysis and direct sequencing. This gene remains a strong candidate for FRDA.

The human DNA polymerase beta gene structure. Evidence of alternative splicing in gene expression.

DNA polymerase beta (beta-pol) is a single-copy gene that is considered to be part of the DNA repair machinery in mammalian cells. Using two human genomic libraries we have cloned the complete human beta-pol gene and determined the organization of the beta-pol coding sequence within the gene. The human beta-pol gene spans 33 kb and contains 14 exons that range from 50 to 233 bp. The 13 introns vary from 96 bp to 6.5 kb. Information derived from this study was used in defining the location of a deletion/insertion type restriction fragment length polymorphism (RFLP) 5' to exon I of the human beta-pol gene. This RFLP was utilized in linkage analysis of DNAs from CEPH families and the results confirm the previous assignment of the human beta-pol gene to chromosome 8 (p12-p11). Analysis of mRNA from six human cell lines using the polymerase chain reaction showed the expression of two beta-pol transcripts. Sequence analysis revealed that the size difference in these transcripts was due to deletion of the 58 bp sequence encoded by exon II, suggesting that the smaller transcript results from an alternative splicing of the exon II sequence during processing of the beta-pol precursor RNA.

Structure of the mouse lysyl oxidase gene.

Lysyl oxidase, an extracellular enzyme involved in the maturation of collagen and elastin, also appears to function as a phenotypic suppressor of transformation by the ras gene product, p21. Genomic clones of the mouse lysyl oxidase gene have been isolated, analyzed, and sequenced. Lysyl oxidase appears to be a single-copy gene, organized into seven exons and six introns, and spans approximately 14 kb of the mouse genome. The gene encodes two messages, sized at about 4.8 and 3.8 kb, that differ in the length of the untranslated sequence at the 3' end of the gene. All of the 3' untranslated sequence and the polyadenylation signals are contained in exon VII; there is no evidence of alternate splicing. Primer extension and ribonuclease protection experiments revealed two sites of transcription initiation in a region with sequence motifs characteristic of a promoter, which was upstream and adjacent to the 5' untranslated sequence found in the cDNA.

Acyl-CoA-binding protein/diazepam-binding inhibitor gene and pseudogenes: A typical housekeeping gene family

Acyl-CoA-binding protein (ACBP) is a 10 kDa protein isolated from bovine liver by virtue of its ability to bind and induce the synthesis of medium-chain acyl-CoA esters. Surprisingly, it turned out to be identical to a protein named diazepam-binding Inhibitor (DBI) claimed to be an endogenous modulator of the GABA A receptor in brain membranes. ACBP/DBI, or proteolytically derived polypeptides of ACBP/DBI, have also been implicated in the control of steroidogenesis in mitochondria and glucose-stimulated insulin secretion. Thus, it appears that ACBP/DBI is a remarkable, versatile protein. Now we have molecularly cloned and characterized the ACBP/DBI gene family in rat. The rat ACBP/DBI gene family comprises one expressed gene and four processed pseudogenes of which one was shown to exist in two allelic forms. The expressed gene is organized into four exons and three introns. There is a remarkable correspondence between the structural modules of ACBP/DBI as determined by 1H nuclear magnetic resonance spectroscopy and the exonintron architecture of the ACBP/DBI gene. Detailed analyses of transcription of the ACBP/DBI gene in brain and liver were performed to map transcription initiation sites and to examine if transcripts from the ACBP/DBI gene were subject to alternative processing. In both brain and liver, transcription is initiated from two major and multiple minor initiation sites. No evidence for alternative splicing was obtained. The promoter region of the ACBP/DBI gene is located in a CpG island and lacks a canonical TATA box. Thus, the ACDB/DBI gene exhibits all the hallmarks of a typical housekeeping gene.

Multiple variants of the human lymphocyte homing receptor CD44 generated by insertions at a single site in the extracellular domain.

The human CD44 cell-surface glycoprotein participates in a wide variety of cell-cell interactions including lymphocyte homing and tumor metastasis. The CD44 antigen is known to display extensive size heterogeneity when compared between different tissue sources although the structural basis for this variation is not yet clear. Recently, two further isotypes in addition to the basic hemopoietic form of the CD44 antigen have been cloned and sequenced and these have been found to contain all or part of a 200-400-base pair insert within the extracellular domain, suggesting that the characteristic heterogeneity in the molecule may be generated by a mechanism of alternative splicing. We have obtained further evidence for alternative splicing, and we report here the cloning and sequencing of six different CD44 sequence variants from a variety of cell lines using a combination of expression cloning and the polymerase chain reaction. Comparison of these variants indicates that each is probably assembled by the insertion of five different exon units in tandem into a discrete site within the membrane proximal region of the extracellular domain. One of the variants contains an exon that shares extensive amino acid sequence homology with a recently described rat CD44 variant that mediates tumor metastasis. Another variant contains a new exon that encodes a tandem repeat of the consensus sequence SG for covalent modification with chondroitin sulfate and is expressed predominantly on mammary tumors. We suggest that a mechanism of alternative exon splicing generates much of the observed structural heterogeneity of CD44 and that the particular set of CD44 variants expressed in a single cell may represent a precise postal code directing the final destination of migrating cells and metastatic tumors.

Rat follistatin: Gonadal and extragonadal expression and evidence for alternative splicing

Follistatin (FS) or FSH-suppressing protein is a polypeptide which exists in multiple forms and has inhibin-like activity. We investigated the distribution of tissues expressing FS mRNA. A segment of the rat FS mRNA corresponding to the last 157 nucleotides of exon 5 and the first 71 nucleotides of exon 6 was prepared by PCR and subcloned in plasmid GEM4Z (pGEM4Z) to produce a radiolabeled RNA probe. S1-Nuclease analysis of RNA extracted from rat tissues indicated alternative splicing of the rat FS gene and gonadal as well as extragonadal expression. In addition to the ovary and testis, the FS mRNA was detected in cerebral cortex, pituitary, adrenal, thymus, pancreas, gut, kidney, heart, uterus, skeletal muscle and lung. Treatment of female rats with combined Pregnant Mare Serum Gonadotropin (PMSG) and human Chorionic Gonadotropin (hCG) increased expression of FS mRNA in the ovary. Our results show that the mRNA for the precursor of the larger known form of FS (FS 344) is much more abundant than the mRNA for the smaller molecule (less than 5%) and that stimulation of FS transcription by PMSG and hCG in the ovary does not change this relationship.

Identification of an additional member of the protein-tyrosine-phosphatase family: evidence for alternative splicing in the tyrosine phosphatase domain.

Protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.13.48) have been implicated in the regulation of cell growth; however, to date few tyrosine phosphatases have been characterized. To identify additional family members, the cDNA for the human tyrosine phosphatase leukocyte common antigen (LCA; CD45) was used to screen, under low stringency, a mouse pre-B-cell cDNA library. Two cDNA clones were isolated and sequence analysis predicts a protein sequence of 793 amino acids. We have named the molecule LRP (LCA-related phosphatase). RNA transfer analysis indicates that the cDNAs were derived from a 3.2-kilobase mRNA. The LRP mRNA is transcribed in a wide variety of tissues. The predicted protein structure can be divided into the following structural features: a short 19-amino acid leader sequence, an exterior domain of 123 amino acids that is predicted to be highly glycosylated, a 24-amino acid membrane-spanning region, and a 627-amino acid cytoplasmic region. The cytoplasmic region contains two approximately 260-amino acid domains, each with homology to the tyrosine phosphatase family. One of the cDNA clones differed in that it had a 108-base-pair insertion that, while preserving the reading frame, would disrupt the first protein-tyrosine-phosphatase domain. Analysis of genomic DNA indicates that the insertion is due to an alternatively spliced exon. LRP appears to be evolutionarily conserved as a putative homologue has been identified in the invertebrate Styela plicata.

Isolation of an active gene encoding human hnRNP protein A1: Evidence for alternative splicing

Heterogeneous nuclear ribonucleoprotein (hnRNP) core protein A1 is a major component of mammalian hnRNP 40 S particles. We describe the structure of an active A1 gene and report on the partial characterization of the A1 gene family. About 30 A1-specific sequences are present per haploid human genome: 15 such sequences were isolated from a human genomic DNA library. Many corresponded to pseudogenes of the processed type but by applying a selection for actively transcribed regions we isolated an active A1 gene. The gene spans a region of 4.6 × 10 3 base-pairs and it is split into ten exons that encode the 320 amino acid residues of the protein. The amino acid sequence derived from the exon sequences is identical with that deduced from cDNA and reported for the protein. One intron exactly separates the two structural domains that constitute the protein. Each of the two RNA-binding domains in protein A1 is encoded by one exon. Experimental evidence indicates that the A1 gene can encode for more than one protein by alternative splicing. The gene is preceded by a strong promoter that contains at least two CCAAT boxes and two possible Sp1 binding sites, but it lacks a TATA box.

CDNA cloning of porcine transforming growth factor-beta 1 mRNAs. Evidence for alternate splicing and polyadenylation.

Most eukaryotic cells encode principally a 2.5-kilobase (kb) transforming growth factor (TGF)-beta 1 mRNA. However, we have found two major TGF-beta 1 RNA species, 3.5 and 2.5 kb long, in porcine tissues. The 3.5-kb species has a longer 3'-untranslated sequence generated by the selection of an alternate polyadenylation site. There is a 117-nucleotide sequence within this unique 3' region, which is similar to the PRE-1 repetitive sequence of unknown function, reported earlier in the porcine genome. We have also cloned and characterized an alternately spliced mRNA species specific for the TGF-beta 1 gene, in which exons IV and V of the corresponding human TGF-beta 1 gene are deleted. The nucleotide sequence of this cDNA clone predicts a putative precursor protein of 256 amino acids; the N-terminal 211 amino acids of this putative protein are identical to the TGF-beta 1 precursor protein (exons I, II, and III of the human TGF-beta 1 gene), but the C-terminal 45 amino acids are distinct, due to a frameshift in the translation of exons VI and VII. In addition we provide data for the existence of other mRNA species generated in a tissue-specific manner either by alternate splicing or by heterogeneous 5' leader sequences.

A novel human cytochrome P450 gene (P450IIB): chromosomal localization and evidence for alternative splicing.

We have isolated from a single human liver cDNA library two clones which are highly homologous (78% over the coding region) to the major phenobarbital-inducible P450 from rat (P450IIB1). This is the first direct demonstration of the presence of the P450IIB gene subfamily in humans. This subfamily is much less extensive than the rodent homologues, but does appear to contain at least two genes. Of the cDNA clones isolated one is apparently normally spliced, whereas the other lacks exon 8 and retains all or part of intron 5. Both clones contain transcribed Alu sequences. The human P450IIB gene has been located to chromosome 19q12----19q13.2 using a probe derived from intron 5, and is close to the CYP 2A locus encoding cytochrome P450IIA2. Restriction fragment length polymorphisms have been found with the enzymes BamHI and MspI which will enable linkage to be determined between these two loci.

Molecular cloning and sequencing of H-2K k cDNA: comparison with other H-2 genes and evidence for alternative splicing

A cDNA library was constructed from mouse L cells that were transfected with a human HLA-B7 gene fragment lacking the 5′-segment of exon 2 and all upstream sequences. A cDNA clone (pESP-C103) which is of mouse origin was detected by hybridization with the HLA-B7 gene. Comparison of the amino acid (aa) sequence predicted from the nucleotide sequence of the cDNA and the partial aa sequence of the H-2k k antigen suggests that this cDNA (pESP-C103) codes for the H-2K k antigen. This cDNA clone extends to the middle of the leader sequence. Comparison of the nucleotide and deduced aa sequences with those of other H-2 genes revealed an alternative splicing in exon 8 in the case of the H-2k k gene.