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PubMed2004

Identification of regulatory cis-acting elements for alternative splicing of presenilin 2 exon 5 under hypoxic stress conditions.

An alternatively spliced form of the presenilin 2 (PS2) gene lacking exon 5 (PS2V) was found in human brains with sporadic Alzheimer's disease. PS2V was induced by hypoxic stress in human neuroblastoma SK-N-SH cells, indicating that hypoxic stress affects the splicing machineries for PS2 exon 5. Here, we identified the critical cis-acting element (sec 2) on the PS2 pre-mRNA responsible for the aberrant splicing of PS2 exon 5 under hypoxic stress conditions. The element was composed of 23 nucleotides in exon 5 and RNA structural analyses showed a stem-loop structure in this sequence. Treatment with an antisense oligonucleotide directed toward the cis-acting element caused an increase in exon 5 inclusion. These results indicate that the sec 2 identified in this study is a novel regulatory element for exon 5 splicing under stress conditions and that trans-acting factors could specifically bind to the element to skip exon 5 of PS2.

Alternative Splicing↗

PubMed1993

Induction of gene expression by exon 2 of the major E1A proteins of adenovirus type 5.

We have constructed an adenovirus type 5 (Ad5) E1A mutant, dl1119/520, that produces essentially only exon 2 of the major E1A proteins. In infected primary baby rat kidney cells, this mutant induced expression of the E1B 55-kDa protein, and in infected human KB cells, it induced expression of this protein, the E2A 72-kDa protein, and hexon. In KB cells, this mutant grew substantially better than Ad5 dl312, which lacks E1A, and as well as Ad5 dl520, an E1A mutant producing only the 243-residue protein. These results suggest that exon 2 of E1A proteins on its own was able to activate gene expression. We also constructed mutants of dl1119/520, containing small deletions in regions of exon 2 that others found to be associated with effects on the properties of E1A transformants. None of these deletions destroyed gene activation completely, indicating that there may be some redundancy among sequences in exon 2 for inducing gene expression. The two deletions that decreased induction the most, residues 224 to 238 and 255 to 270, were in regions reported to be associated with the expression of a metalloprotease and with enhanced transformation, suggesting that exon 2 may regulate expression of genes governing cell growth. It is remarkable that all sections of E1A proteins, exon 1, the unique region, and exon 2, have now been found to affect gene expression.

Adenoviridae↗

PubMed1995

Epstein-Barr virus latent messages with shuffled leader exons: remnants of circumgenomic transcription?

The EBNA transcription unit which is active in Epstein-Barr virus-immortalized latently infected B lymphocytes covers approximately 60% of the 172-kb genome. Since the genome exists as a circular double-stranded DNA molecule in latently infected cells, it is conceivable that complete copies are made during transcription. Rather than attempt to detect gigantic RNA molecules directly, we used RNA-PCR to detect incorporation of leader exons into mRNA in a shuffled order. The downstream U leader exon was detected spliced upstream of the internal repeat leader exons W1 and W2 in the polyadenylated RNA fraction of spontaneous lymphoblastoid cell lines, restricted phenotype BL cell lines Wanyanyi and Wewak2, and in B95-8, Raji, and Akata cells. Quantitative competitive RNA-PCR showed that the ratio of U exon-containing EBNA1 messages to U exon-shuffled leader messages was approximately 10:1, with large variation from cell line to cell line, and was not affected by induction of the lytic cycle in B95-8, Raji, or Akata cells. Messages with shuffled exons contained only the C2W1 alternative splice, which does not produce an initiator AUG methionine codon for EBNA4 gene expression. The results provide evidence for long-range exon skipping and imply that genome-length transcripts may occur and participate in viral gene expression in latency.

Adult↗

PubMed1994

Two different sequence elements within exon 4 are necessary for calcitonin-specific splicing of the human calcitonin/calcitonin gene-related peptide I pre-mRNA.

The calcitonin (CT)/calcitonin gene-related peptide I (CGRP-I) gene (CALC-I gene) is subject to alternative tissue-specific processing of its primary transcript. CT mRNA is the predominant mRNA produced in thyroid C cells, whereas CT gene-related peptide I mRNA is the main product in neurons of the central and peripheral nervous systems. The CT-specific exon 4 is surrounded by weak processing sites. In this study we have investigated whether exon 4 sequences are involved in the tissue-specific selection of the exon 4 splice acceptor site. The results indicate that two separate elements, termed A and B, in the 5' part of exon 4 are required for production of CT-specific RNA. These sequences are located between nucleotides 67 and 88 (A) and nucleotides 117 and 146 (B) relative to the 5' end of exon 4. Variation of the distance between these sequence elements and the 3' splice site of exon 4 does not change the processing choice. These sequence elements are functionally equivalent. CT-specific splicing requires the presence of both sequence A and B or duplicates of either sequence element in exon 4. The effect of these sequences on the RNA processing choice is overruled by mutation of the CT-specific uridine branch acceptor nucleotide into a commonly preferred adenosine residue.

Alternative Splicing↗

PubMed1996

Exon 2-mediated c-myc mRNA decay in vivo is independent of its translation.

We have previously shown that the steady-state level of c-myc mRNA in vivo is primarily controlled by posttranscriptional regulatory mechanisms. To identify the sequences involved in this process, we constructed a series of H-2/myc transgenic lines in which various regions of the human c-MYC gene were placed under the control of the quasi-ubiquitous H-2K class I regulatory sequences. We demonstrated that the presence of one of the two coding exons, exon 2 or exon 3, is sufficient to confer a level of expression of transgene mRNA similar to that of endogenous c-myc in various adult tissues as well as after partial hepatectomy or after protein synthesis inhibition. We now focus on the molecular mechanisms involved in modulation of expression of mRNAs containing c-myc exon 2 sequences, with special emphasis on the coupling between translation and c-myc mRNA turnover. We have undertaken an analysis of expression, both at the mRNA level and at the protein level, of new transgenic constructs in which the translation is impaired either by disruption of the initiation codon or by addition of stop codons upstream of exon 2. Our results show that the translation of c-myc exon 2 is not required for regulated expression of the transgene in the different situations analyzed, and therefore they indicate that the mRNA destabilizing function of exon 2 is independent of translation by ribosomes. Our investigations also reveal that, in the thymus, some H-2/myc transgenes express high levels of mRNA but low levels of protein. Besides the fact that these results suggest the existence of tissue-specific mechanisms that control c-myc translatability in vivo, they also bring another indication of the uncoupling of c-myc mRNA translation and degradation.

Animals↗

PubMed1997

Cloning of Caenorhabditis U2AF65: an alternatively spliced RNA containing a novel exon.

The U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF, is an essential splicing factor required for recognition of the polypyrimidine tract and subsequent U2 snRNP assembly at the branch point. Because Caenorhabditis elegans introns lack both polypyrimidine tract and branch point consensus sequences but have a very highly conserved UUUUCAG/R consensus at their 3' splice sites, we hypothesized that U2AF might serve to recognize this sequence and thus promote intron recognition in C. elegans. Here we report the cloning of the gene for the large subunit of U2AF, uaf-1. Three classes of cDNA were identified. In the most abundant class the open reading frame is similar to that for the U2AF65 from mammals and flies. The remaining two classes result from an alternative splicing event in which an exon containing an in-frame stop codon is inserted near the beginning of the second RNA recognition motif. However, this alternative mRNA is apparently not translated. Interestingly, the inserted exon contains 10 matches to the 3' splice site consensus. To determine whether this feature is conserved, we sequenced uaf-1 from the related nematode Caenorhabditis briggsae. It is composed of six exons, including an alternatively spliced third exon interrupting the gene at the same location as in C. elegans. uaf-1 is contained in an operon with the rab-18 gene in both species. Although the alternative exons from the two species are not highly conserved and would not encode related polypeptides, the C. briggsae alternative exon has 18 matches to the 3' splice site consensus. We hypothesize that the array of 3' splice site-like sequences in the pre-mRNA and alternatively spliced exon may have a regulatory role. The alternatively spliced RNA accumulates at high levels following starvation, suggesting that this RNA may represent an adaption for reducing U2AF65 levels when pre-mRNA levels are low.

Alternative Splicing↗

PubMed1999

A premature termination codon interferes with the nuclear function of an exon splicing enhancer in an open reading frame-dependent manner.

Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5' element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5' and 3' elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3' enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3' splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.

Animals↗

PubMed2002

Detection of exon polymorphisms in the human lactoferrin gene.

We previously demonstrated that lactoferrin gene polymorphisms occur in cancer cells of patients with leukemia and breast cancer. In this study, we established a non-radioactive polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis, one of the most sensitive and simplest methods to detect polymorphisms and mutations of the human lactoferrin gene. We optimized the PCR conditions for nine different DNA templates and 16 pairs of exon primers for SSCP analysis. The DNA templates used in the analyses were prepared from a cosmid clone (CT6-1) that contains the human lactoferrin gene, human placental tissue, leukocytes from 10 normal volunteers, leukemic cells of two patients, and previously established three breast and two leukemic cell lines. With the appropriate exon-primer sets, PCR products from exon I to exon 16 of the lactoferrin gene were generated from the DNA templates and analyzed by SSCP. Compared with the homogenous cloned DNA, lactoferrin gene polymorphisms were detected within exons 2, 5, 7, 9, 13, 14, and 15 of the normal placental and leukocyte DNA. In addition, abnormal migration patterns of the lactoferrin gene in cancer cells were detected in exons 4, 5, 13, 14, and 15. The PCR-SSCP band migration patterns can be attributed either to gene polymorphism in normal cells or to DNA mutations in cancer cells and the employed method cannot distinguish between them. Nonetheless, the present analysis suggests that genetic polymorphisms of the lactoferrin gene exist in selected exons and additional mutations of the lactoferrin gene do occur in the cancer cells.

Breast Neoplasms↗

PubMed2002

Exon truncation by alternative splicing of murine ICAM-1.

The murine gene for intercellular adhesion molecule-1 (ICAM-1) encodes multiple products, arising from alternative splicing. Full-length ICAM-1 contains five extracellular Ig domains, each encoded by a separate exon. Alternatively spliced forms have Ig domains 2, 3, and/or 4 excised as a result of exon skipping. We report here a novel splice variant of murine ICAM-1, resulting from exon truncation rather than exon skipping and affecting Ig domain 5. A 5' splice donor site within exon 6 generates transcripts missing 69 nucleic acids from the 3' terminus of the exon. This in-frame exon truncation is predicted to replace 24 amino acids within Ig domain 5 with a single aspartic acid residue, yielding a structure other than an Ig domain immediately external to the membrane. Expression of this alternatively spliced form is induced in mouse lungs, spleen, and kidneys during LPS-induced pulmonary inflammation. Since the affected region is critical for ICAM-1 presentation, dimerization, and solubilization, this alternative splice variant may have unique physiological functions.

Alternative Splicing↗

PubMed2002

Novel 5' exon of scavenger receptor CD36 is expressed in cultured human vascular smooth muscle cells and atherosclerotic plaques.

CD36, a member of the scavenger receptor family, is centrally involved in the uptake of oxidized low density lipoproteins (oxLDLs) from the bloodstream. During the atherosclerotic process, the lipid cargo of oxLDL accumulates in macrophages and smooth muscle cells (SMCs), inducing their pathological conversion to foam cells. Increased expression of CD36 occurs in human atherosclerotic lesions, and CD36 knockout mice show reduced uptake of modified LDLs and reduced atherosclerosis. Here, we describe a novel exon 1b and extended CD36 promoter in human SMCs. Exon 1b is specifically transcribed in activated aortic SMCs and mainly expressed in atherosclerotic plaques. Thus, switching to exon 1b transcription may be an important step for the activation of SMCs and their conversion to foam cells. Using an antisense oligonucleotide to exon 1b, we inhibit CD36 translation and highly reduce oxLDL uptake. The antisense to exon 1b does not affect CD36 in cell lines not expressing the new exon. The possibility of a novel antiatherosclerotic therapy and the use of exon 1b as a marker of atherosclerosis are discussed.

5' Flanking Region↗

PubMed2006

High frequency of BMPR2 exonic deletions/duplications in familial pulmonary arterial hypertension.

RATIONALE: Previous studies have shown that approximately 55% of patients with familial pulmonary arterial hypertension (FPAH) have BMPR2 coding sequence mutations. However, direct sequencing does not detect other types of heterozygous mutations, such as exonic deletions/duplications. OBJECTIVE: To estimate the frequency of BMPR2 exonic deletions/duplications in FPAH. METHODS: BMPR2 mRNA from lymphoblastoid cell lines of 30 families with PAH and 14 patients with idiopathic PAH (IPAH) was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing. Sequencing of genomic DNA was used to identify point mutations in splice donor/acceptor sites. Multiplex ligation-dependent probe amplification (MLPA) was used to detect exonic deletions/duplications with verification by real-time PCR. MEASUREMENTS AND MAIN RESULTS: Eleven (37%) patients with FPAH had abnormally sized RT-PCR products. Four of the 11 patients were found to have splice-site mutations resulting in aberrant splicing, and exonic deletions/duplications were detected in the remaining seven patients. MLPA identified three deletions/duplications that were not detectable by RT-PCR. Coding sequence point mutations were identified in 11 of 30 (37%) patients. Mutations were identified in 21 of 30 (70%) patients with FPAH, with 10 of 21 mutations (48%) being exonic deletions/duplications. Two of 14 (14%) patients with IPAH exhibited BMPR2 point mutations, whereas none showed exonic deletions/duplications. CONCLUSIONS: Our study indicates that BMPR2 exonic deletions/duplications in patients with FPAH account for a significant proportion of mutations (48%) that until now have not been screened for. Because the complementary approach used in this study is rapid and cost effective, screening for BMPR2 deletions/duplications by MLPA and real-time PCR should accompany direct sequencing in all PAH testing.

Bone Morphogenetic Protein Receptors, Type II↗

PubMed1989

Sequence and exon-intron organization of the DNA encoding the alpha I domain of human spectrin. Application to the study of mutations causing hereditary elliptocytosis.

We have determined the exon-intron organization and the nucleotide sequence of the exons and their flanking intronic DNA in cloned genomic DNA that encodes the first 526 amino acids of the alpha I domain of the human red cell spectrin polypeptide chain. From the gene sequence we designed oligonucleotide primers to use in the polymerase chain reaction technique to amplify the appropriate exons in DNA from individuals with three variants of hereditary elliptocytosis characterized by the presence of abnormal alpha I spectrin peptides, 46-50 and 65-68 kD in size, in partial tryptic digests of spectrin. The alpha I/68-kD abnormality resulted from a duplication of leucine codon 148 in exon 4: TTG-CTG to TTG-TTG-CTG. The alpha I/50a defect was associated in different individuals with two separate single base changes in exon 6: CTG to CCG (leucine to proline) encoding residue 254, and TCC to CCC (serine to proline) encoding residue 255. In another individual with the alpha I/50a polypeptide defect, the nucleotide sequence encoding amino acid residues 221 through 264 was normal. The alpha I/50b abnormality resulted from a single base change of CAG (glutamine) to CCG (proline) encoding residue 465 in exon 11 in two unrelated individuals. In a third individual with alpha I/50b-kD hereditary elliptocytosis, the entire exon encoding residues 445 through 490 was normal. The relationship of the alpha I domain polypeptide structure to these mutations and the organization of the gene is discussed.

Amino Acid Sequence↗

PubMed2006

Origin, splicing, and expression of rodent amelogenin exon 8.

Amelogenin RNA transcripts undergo extensive alternative splicing, and MMP-20 processes the isoforms following their secretion. Since amelogenins have been ascribed cell-signaling activities, we asked if a lack of proteolytic processing by MMP-20 affects amelogenin signaling and consequently alters amelogenin splice site selection. RT-PCR analyses of amelogenin mRNA between control and Mmp20(-/-)mice revealed no differences in the splicing pattern. We characterized 3 previously unidentified amelogenin alternatively spliced transcripts and demonstrated that exon-8-encoded amelogenin isoforms are processed by MMP-20. Transcripts with exon 8 were expressed approximately five-fold less than those with exon 7. Analyses of the mouse and rat amelogenin gene structures confirmed that exon 8 arose in a duplication of exons 4 through 5, with translocation of the copy downstream of exon 7. No downstream genomic sequences homologous to exons 4-5 were present in the bovine or human amelogenin genes, suggesting that this translocation occurred only in rodents.

Alternative Splicing↗

PubMed2006

Assessing the number of ancestral alternatively spliced exons in the human genome.

BACKGROUND: It is estimated that between 35% and 74% of all human genes undergo alternative splicing. However, as a gene that undergoes alternative splicing can have between one and dozens of alternative exons, the number of alternatively spliced genes by itself is not informative enough. An additional parameter, which was not addressed so far, is therefore the number of human exons that undergo alternative splicing. We have previously described an accurate machine-learning method allowing the detection of conserved alternatively spliced exons without using ESTs, which relies on specific features of the exon and its genomic vicinity that distinguish alternatively spliced exons from constitutive ones. RESULTS: In this study we use the above-described approach to calculate that 7.2% (+/- 1.1%) of all human exons that are conserved in mouse are alternatively spliced in both species. CONCLUSION: This number is the first estimation for the extent of ancestral alternatively spliced exons in the human genome.

Algorithms↗

PubMed2025

Rescue of common and rare exon 2 skipping variants of the GAA gene using modified U1 snRNA.

BACKGROUND: Pompe disease (PD) is an autosomal recessive lysosomal storage disorder caused by the deficient activity of acid alpha glucosidase (GAA) enzyme due to mutations in the GAA gene. As a result, undigested glycogen accumulates within lysosomes causing their dysfunction. From a clinical point of view, the disease can be classified in infantile-onset (IO) and late-onset (LO) forms. The common GAA c.-32-13T>G variant, found in 40-70% of LO-PD alleles, is a leaky splicing mutation interfering with the correct GAA exon 2 recognition by the spliceosome leading to the production of non-functional GAA transcripts. In this study, we used modified, GAA-tailored U1 snRNAs to correct the aberrant splicing determined by the c.-32-13T>G and other GAA exon 2-skipping mutations. METHODS: A set of constructs expressing 5 different engineered U1 snRNAs was generated. A functional splicing assay using a GAA hybrid minigene carrying different variants known to affect GAA exon 2 splicing was used to test the effect of engineered U1 snRNAs on exon 2 inclusion. The effect on endogenously expressed GAA transcript and GAA enzymatic activity was assessed by transfecting patient-derived fibroblasts bearing the common c.-32-13T>G with the best performing modified U1 snRNA. RESULTS: Modified U1-3, U1+1 and U1+6 snRNAs were all able to increase, in a dose-dependent manner, the inclusion of exon 2 within the transcript derived from the GAA minigene harbouring the c.-32-13T>G variant. The U1+1 was the most effective one (2,5 fold increase). Moreover, U1+1 snRNA partially rescued the correct splicing of GAA minigenes harbouring mutations that affect the 3'ss (c.-32-3C>G, c.-32-2A>G) and the 5'ss (c.546G>A, c.546G>C, c.546G>T). Notably, the treatment of patient-derived fibroblasts carrying the c.-32-13T>G mutation with the U1+1 snRNA increased the amount of normal GAA mRNA by 1,8 fold and the GAA enzymatic activity by 70%. CONCLUSIONS: we provide the proof-of-concept for the use of modified GAA-tailored U1 snRNAs, designed to potentiate the recognition of the GAA exon 2 5'ss, as therapeutic tools to correct the aberrant transcripts carrying variants that affect exon 2 splicing, including the common c.-32-13T>G variant.

Humans↗

PubMed1997

Cloning and functional expression of the luteinizing hormone receptor complementary deoxyribonucleic acid from the marmoset monkey testis: absence of sequences encoding exon 10 in other species.

Based on sequence homologies among the human, porcine, rat, and mouse genes for the LH receptor (LHR), overlapping partial fragments of LHR complementary DNAs (cDNAs) were multiplied from marmoset monkey testicular RNA using reverse transcription-PCR. Ligations of the individual cDNA fragments generated a full-length monkey LHR cDNA (2031 bp) containing the complete amino acid-coding sequence (676 amino acids). Northern hybridization analysis of monkey testicular RNA, using a complementary RNA probe corresponding to the full-length cDNA, demonstrated major transcripts of 5.5 and 1.4 kilobases and minor ones of 4.0, 2.7, and 1.9 kilobases. Sequence analysis of the monkey LHR cDNA revealed a striking feature, i.e. the absence of an 81-bp nucleotide sequence corresponding to exon 10, present in the LHR cDNAs of all other species studied to date. The monkey LHR cDNA displayed 83-94% overall sequence homology with the other mammalian LHR cDNAs. Reverse transcription-PCR with human exon 10-specific primers demonstrated the total absence of this sequence from the monkey LHR messenger RNA. Southern hybridization of monkey genomic DNA using a human exon 10 probe demonstrated its presence in the monkey gene and that it is totally spliced out from the primary transcript. COS cells transfected with the monkey LHR cDNA showed similar high affinity (Kd = 0.25 nmol/liter) of [125I]iodo-hCG binding as those transfected with human LHR cDNA (Kd = 0.20 nmol/liter). The cells expressing the recombinant monkey and human LHR displayed similar responses of extracellular cAMP and inositol trisphosphate to hCG. In conclusion, marmoset monkey LHR seems to lack the sequence corresponding to exon 10 of the LHR gene in other mammalian species. The truncation does not alter LHR function, as the monkey receptor protein bound hCG and evoked cAMP and inositol trisphosphate responses comparable to those of the human LHR containing the exon 10-encoded structure. As the sequence homologous to exon 10 is missing in the other two glycoprotein receptors, i.e. those of FSH and TSH, this extra exon is apparently inserted into the LHR messenger RNA of some species during evolution from intronic sequences by a change in alternative splicing.

Alternative Splicing↗

PubMed1996

Expression of exon 3-retaining and -deleted human growth hormone receptor messenger ribonucleic acid isoforms during development.

Recent investigations have suggested that human GH (hGH) and its receptor may have specific functions during human fetal life. To improve our understanding of the mechanisms of hGH action during gestation, we characterized the ontogenic appearance of hGH receptor messenger ribonucleic acid (mRNA) in multiple human fetal and postnatal tissues. Using RT-PCR assays, followed by Southern hybridization to confirm the specificity of the amplified fragments, we scanned the entire coding region of the hGH receptor mRNA. Transcription of the hGH receptor gene was observed in all fetal tissues studied (liver, kidney, skin, muscle, lung, adrenal, spleen, intestine, central nervous system, pancreas, and placental villi) from the earliest stage that could be examined [7-14.8 weeks fetal age (FA)]. Furthermore, we identified only 2 isoforms of the hGH receptor mRNA-coding region: exon 3 can be retained or deleted. Surprisingly, we found individual-specific, not tissue-specific, expression patterns of these two transcripts when we examined multiple tissues (n = 2-6) from 15 individuals (11.5-33 weeks FA); this individual-specific pattern of expression is maintained in cultured dermal fibroblasts for at least 12 generations (n = 2; 16 and 20 weeks FA). In addition, a cross-sectional study of 78 individuals (9 weeks FA to 43 yr postnatal age) showed that the exon 3-deleted transcript is predominantly expressed in tissues from fetuses of 9-20 weeks FA (P < 0.002). Finally, we showed that the absence of exon 3 from the mRNA is not due to genomic deletion of exon 3 by amplifying exon 3 from genomic DNA of 3 fetuses (13.3-19 weeks FA) expressing only the exon 3-deleted mRNA transcript. We conclude that 1) transcription of the hGH receptor gene occurs in multiple tissues as early as the first trimester of human fetal life; 2) the exon 3-retaining and -deleted transcripts are the only two isoforms of the hGH receptor mRNA-coding region during gestation; and 3) the pattern of expression of these transcripts is individual specific and may be developmentally regulated.

Base Sequence↗

PubMed2005

Identification of two further splice variants of GABABR1 characterizes the conserved micro-exon 4 as a hot spot for regulated splicing in the rat brain.

Inhibitory neurotransmission in the mammalian brain is principally mediated by gamma-aminobutyric acid (GABA) acting through different subtypes of cell membrane GABA receptor (GABAR). The expression of one GABAR gene, GABABR1, is distinguished by the expression of multiple splice variants that encode different isoforms of the receptor. In the present study, we have identified two novel GABABR1 variants, GABABR1h (R1h) and GABABR1i (R1i), which appear to arise from alternative splicing of the GABABR1 gene. The expression of R1h and R1i is differentially regulated in brain and peripheral tissues, but expression is not altered in the brain of a genetic model of absence epilepsy (GAERS rat [genetic absence epilepsy rat from Strasbourg]). Both the R1h and R1i variants exhibit a novel 80-bp insert downstream of exon 4 that is flanked by consensus splice sites, and both encode C-terminal-truncated proteins. The new insight into the family of GABABR1 variants gained from this study identifies exon 4 as a preferred locus, or hot spot for regulated splicing in the GABABR1 gene. This finding correlates with the micro-exonic nature of exon 4 (21 bp). Bioinformatic analysis of micro-exon 4 and its flanking pre-mRNA sequences has revealed multiple, potentially competitive, exonic splicing enhancers that provide a mechanistic basis for the preponderance of alternative splicing events at this locus. Conservation of GABABR1 micro-exon 4 across species suggests a conserved functional role, facilitating either N-terminal protein production or post-transcriptional gene regulation through regulated splicing coupled to transcript decay.

Alternative Splicing↗