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At least 73 records · Page 4Linked to original sources
PubMed2003

Lack of mu-opioid receptor-mediated G-protein activation in the spinal cord of mice lacking Exon 1 or Exons 2 and 3 of the MOR-1 gene.

The G-protein activation induced by mu-opioid receptor agonists was determined in spinal cord membranes from two types of mu-opioid receptor knockout mice: mice with a disruption of exon 1 (MOR (Exon 1)-KO) or exons 2 and 3 (MOR (Exons 2 and 3)-KO) of the mu-opioid receptor gene. The G-protein activation induced by the opioid agonists was measured by monitoring the increases of guanosine-5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding. The mu-opioid receptor agonists (D-Ala(2),N-MePhe (4),Gly-ol(5)]enkephalin, endomorphin-1, endomorphin-2, morphine, morphine-6 beta-glucuronide, and fentanyl produced concentration-dependent increases of [(35)S]GTP gamma S binding to spinal cord membranes in wild-type mice, but not in MOR (Exon 1)-KO mice or MOR (Exons 2 and 3)-KO mice. On the other hand, the delta-opioid receptor agonist [D-Pen (2,5)]enkephalin, the kappa-opioid receptor agonist (-)U50,488H, or the ORL1-receptor agonist nociception increased [(35)S]GTP gamma S binding in the spinal cord membranes from both MOR (Exon 1)-KO mice and MOR (Exons 2 and 3)-KO mice to the same extent as in the corresponding wild-type mice. The results provide further information about the important roles of the sequences encoded within exon 1 and exons 2 and 3 of mu-opioid receptor gene for the activation of G-proteins by mu-opioid receptor agonists in the mouse spinal cord.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed2000

Analysis of the CC chemokine receptor 3 gene reveals a complex 5' exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements.

To understand the regulation of CC chemokine receptor 3 (CCR3) expression, its gene structure and promoter have been characterized. The CCR3 gene contains 4 exons that give rise to multiple messenger RNA (mRNA) species by alternative splicing. Exon 1 is present in all transcripts, whereas exon 2 or 3 is present at low frequency (< 10%). Exon 4 contains the open reading frame and 11 bp of the 5' untranslated region. Northern analysis revealed 4 species of CCR3 mRNA. Direct sequencing revealed that the first 1 kb of the promoter and exon 1 contained only one mutation in 19 individuals, indicating that the CCR3 promoter and exon 1 are conserved between individuals. The first 1.6 kb of the 5' flanking region of exon 1 contained promoter elements including a TATA box and motifs for myeloid transcription factors and had strong promoter activity in eosinophilic, lymphoid, myeloid, and respiratory epithelial cell lines. Deletion analysis revealed differential regulation of the CCR3 promoter in eosinophilic and epithelial cells suggesting the presence of lineage-specific elements. Interestingly, exon 1 enhanced the activity of the promoter and this effect was especially prominent in eosinophilic cells. Thus, the human CCR3 gene has a complex 5' exon structure, a conserved promoter with strong activity in multiple cell types, and a functional 5' untranslated exon.

Alternative Splicing↗
PubMed1995

Multiple APC messenger RNA isoforms encoding exon 15 short open reading frames are expressed in the context of a novel exon 10A-derived sequence.

Intragenic splice mechanisms affecting the coding exons 8 to 15 of the human adenomatous polyposis coli (APC) gene have been analyzed. Three mechanisms within this gene area were found to contribute to mRNA heterogeneity: (i) facultative expression of exon 9-encoded sequences; (ii) in-frame insertion of a 54-nucleotide sequence encoded by a novel exon located 1.6 kb down-stream from exon 10, provisionally designated APC exon 10A; (iii) skipping of exon 14, resulting in a novel exon 13/15 connection. Interestingly the latter event provided the mRNA with a novel open reading frame, which was terminated after 19 codons of exon 15-derived sequences. Combinatorial joining of these segments yielded 7 different transcripts in addition to an mRNA species resulting from an exon 10/15 connection, as determined by cloning and sequence analysis. RT-PCR expression analyses were carried out to demonstrate that this complexity of splice variants is indeed synthesized in cell lines derived from various tissues. Furthermore, in accordance with our findings at the transcript level, we provide Western blot analyses demonstrating that moderate steady-state levels of genuine APC-specific low m.w. polypeptide chains exist. These APC "light chains", however, are not identical with polypeptide chains, which have been reported to accompany apoptosis and necrosis, since the molecules described here are definitively co-expressed with p300apc at the transcript and protein levels.

Amino Acid Sequence↗
PubMed2006

Exploring the frontiers of therapeutic exon skipping for Duchenne muscular dystrophy by double targeting within one or multiple exons.

Through antisense-induced single-, double-, and multiexon skipping, we have previously demonstrated restoration of dystrophin expression in Duchenne muscular dystrophy (DMD) patient-derived muscle cells in vitro. In this study we further explored the frontiers of this strategy by using specific combinations of 2'-O-methyl phosphorothioate antisense oligonucleotides (AONs) targeting either one or multiple exons. We show that skipping efficiencies may indeed be improved by targeting two putative splicing regulatory sequences within one exon. In particular, such double targeting was effective for the thus far "unskippable" exons 47 and 57. We previously reported the feasibility of multiexon skipping spanning exon 45 to exon 51, using a combination of AONs targeting both outer exons (45 and 51). This would be applicable to 13% of all DMD patients. We here explored the frontiers of multiexon skipping both to increase the number of patients that can be treated with the same set of AONs and to mimic large deletions found in relatively mildly affected BMD patients. We aimed at inducing larger multiexon-skipping stretches, such as exons 17-51, exons 42-55, and exons 45-59. However, this appeared complicated and may be dependent on cotranscriptional splicing and the size of the flanking introns.

Cells, Cultured↗
PubMed2000

The splicing determinants of a regulated exon in the axonal MAP tau reside within the exon and in its upstream intron.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exon 6 of the gene is an alternatively spliced cassette whose expression profile is distinct from that of the other tau regulated exons, implying the utilization of distinct regulatory factors. Previous work had established the use of cryptic splice sites within exon 6 and the influence of flanking exons on the ratio of exon 6 variants. The present work shows that, in addition to the previously identified participants, the ratio of exon 6 isoforms is affected by: (1) suppression of the cryptic sites, (2) deletions of the upstream intron, and (3) the splicing regulators PTB and U2AF, both of which act on the branch point/polypyrimidine tract region. These results strongly suggest that factors binding immediately upstream of exon 6 are involved in regulation of this exon. They also lead to the conclusion that splicing of exon 6 is primarily governed by multiple branch points.

Animals↗
PubMed1997

Novel exonic elements that modulate splicing of the human fibronectin EDA exon.

Three exons in the fibronectin primary transcript are alternatively spliced in a tissue- and developmental stage-specific manner. One of these exons, EDA, has been shown previously by others to contain two splicing regulatory elements between 155 and 180 nucleotides downstream of the 3'-splice site: an exon splicing enhancer and a negative element. By transient expression of a chimeric beta-globin/fibronectin EDA intron in COS-7 cells, we have identified two additional exonic splicing regulatory elements. RNA generated by a construct containing the first 120 nucleotides of the fibronectin EDA exon was spliced with an efficiency of approximately 50%. Deletion of most of the fibronectin EDA exon sequences resulted in a 20-fold increase in the amount of spliced RNA, indicative of an exon splicing silencer. Deletion and mutagenesis studies suggest that the fibronectin exon splicing silencer is associated with a conserved RNA secondary structure. In addition, sequences between nucleotides 93 and 118 of the EDA exon contain a non-purine-rich splicing enhancer as demonstrated by its ability to function in a heterologous context.

Alternative Splicing↗
PubMed1998

The exon structure of the human MAGP-2 gene. Similarity with the MAGP-1 gene is confined to two exons encoding a cysteine-rich region.

A cDNA for human microfibril-associated glycoprotein-2 (MAGP-2) was used to screen a human leukocyte genomic DNA library in EMBL-3 vector. One clone, clone H (10 kilobase pairs (kbp)), was isolated that contained most of the MAGP-2 gene. The remainder of the 3' end of the gene was obtained by direct polymerase chain reaction amplification of genomic DNA. The human MAGP-2 gene was found to be about 11 kbp in size and to contain 10 evenly distributed exons. The internal exons range in size from 30 base pairs (bp) to 88 bp with exons 4 and 6 the only exons of equal size (45 bp). All internal intron:exon junctions are defined by canonical splice donor and acceptor sites. Each junction has a 1/2 codon split with the exception of the exon 8/9 junction, which has a 2/1 split. The translation initiation codon is in exon 2, and the final exon contains 110 bp of coding sequence, including 2 cysteine codons. Primer extension experiments identified only one major transcription initiation site, 213 bases upstream of the ATG site. Rapid analysis of cDNA ends-polymerase chain reaction analysis of the 5' end of MAGP-2 mRNA from placenta confirmed this result and did not detect any alternative splicing of transcripts. The putative promoter region of the MAGP-2 gene was found to be AT-rich and it lacked a TATA box and other common regulatory elements. However the sequence surrounding the transcription start site CTCA(+1)TTCC was similar to the consensus CTCA(+1)NTCT (N is any nucleoside) for an initiator element found in terminal deoxynucleotidyltransferase and a number of other highly regulated genes. Comparison with the previously characterized human MAGP-1 gene showed that structural similarity was largely confined to the exact size, sequence, and junction alignment of the two penultimate exons which encode the first six of the seven cysteine residues that are precisely spaced in both proteins. The findings are consistent with the growing evidence that, although MAGP-1 and MAGP-2 are both intimately involved in the biology of fibrillin-containing microfibrils, the MAGPs are structurally, functionally, and developmentally diverse proteins which share one characteristic cysteine-rich motif.

Amino Acid Sequence↗
PubMed1992

Identification of exon sequences and an exon binding protein involved in alternative RNA splicing of calcitonin/CGRP.

Transcripts derived from the 6 exon CALC I gene are differentially processed in a tissue-specific fashion to include or exclude a calcitonin-specific exon 4. All cell types which transcribe a second calcitonin/CGRP gene, CALC II, exclude exon 4. Substitution of the first 30 nucleotides of CALC I exon 4 with analogous CALC II sequence was sufficient to prevent recognition of exon 4 in in vitro or in vivo RNA splicing systems. UV crosslinking detected a approximately 66 kDa RNA-binding protein in HeLa nuclear extract which interacted with CALC I proximal exon sequence, but not CALC II or mutant sequences. UV crosslinking of this protein was inhibited by addition of nuclear extract from a cell type which normally causes exclusion of exon 4. These results identify an important regulatory element within exon 4 and support a model in which calcitonin production requires protein interaction with this sequence to facilitate exon recognition.

Amino Acid Sequence↗
PubMed1995

Relative exon affinities and suboptimal splice site signals lead to non-equivalence of two cassette exons.

Tau is a microtubule-associated protein whose transcript undergoes complex regulated splicing in the mammalian nervous system. Exons 2 and 3 of the gene are alternatively spliced cassettes in which exon 3 never appears independently of exon 2. Expression of tau minigene constructs in cells indicate that exon 2 resembles a constitutive exon, while a suboptimal branch point connected to exon 3 inhibits inclusion of exon 3 in the mRNA. Splicing of the two tau exons is controlled by their relative affinities for each other versus the affinities of their flanking exons for them.

Alternative Splicing↗
PubMed1996

Scanning for genes in large genomic regions: cosmid-based exon trapping of multiple exons in a single product.

To facilitate the scanning of large genomic regions for the presence of exonic gene segments we have constructed a cosmid-based exon trap vector. The vector serves a dual purpose since it is also suitable for contig construction and physical mapping. The exon trap cassette of vector sCOGH1 consists of the human growth hormone gene driven by the mouse mettallothionein-1 promoter. Inserts are cloned in the multicloning site located in intron 2 of the hGH gene. The efficiency of the system is demonstrated with cosmids containing multiple exons of the Duchenne Muscular Dystrophy gene. All exons present in the inserts were successfully retrieved and no cryptic products were detected. Up to seven exons were isolated simultaneously in a single spliced product. The system has been extended by a transcription-translation-test protocol to determine the presence of large open reading frames in the trapped products, using a combination of tailed PCR primers directing protein synthesis in three different reading frames, followed by in vitro transcription-translation. Having larger stretches of coding sequence in a single exon trap product rather than small single exons greatly facilitates further analysis of potential genes and offers new possibilities for direct mutation analysis of exon trap material.

Animals↗
PubMed1995

A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon.

Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this report, we examined alternative splicing of a 1-kb internal exon from the human caldesmon gene containing two regulated 5' splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon RNA via utilization of the exon-internal 5' splice site, inclusion of the differential exon required a long purine-rich sequence located between the two competing 5' splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicing enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5' splice site, and functioned in a heterologous exon dependent on exon enhancers for inclusion, indicating that the caldesmon purine-rich sequence can be classified as an ESE. The ESE was required for utilization of the internal 5' splice site only in the presence of the competing 5' splice site and had no effect when placed downstream of the terminal 5' splice site. In the absence of the internal 5' splice site, the ESE activated a normally silent cryptic 5' splice site near the natural internal 5' splice site, indicating that the ESE stimulates upstream 5' splice site selection. We propose that the caldesmon ESE functions to regulate competition between two 5' splice sites within a differential internal exon.

Alternative Splicing↗
PubMed2005

Exon inclusion is dependent on predictable exonic splicing enhancers.

We have previously formulated a list of approximately 2,000 RNA octamers as putative exonic splicing enhancers (PESEs) based on a statistical comparison of human exonic and nonexonic sequences (X. H. Zhang and L. A. Chasin, Genes Dev. 18:1241-1250, 2004). When inserted into a poorly spliced test exon, all eight tested octamers stimulated splicing, a result consistent with their identification as exonic splicing enhancers (ESEs). Here we present a much more stringent test of the validity of this list of PESEs. Twenty-two naturally occurring examples of nonoverlapping PESEs or PESE clusters were identified in six mammalian exons; five of the six exons tested are constitutively spliced. Each of the 22 individual PESEs or PESE clusters was disrupted by site-directed mutagenesis, usually by a single-base substitution. Eighteen of the 22 disruptions (82%) resulted in decreased splicing efficiency. In contrast, 24 control mutations had little or no effect on splicing. This high rate of success suggests that most PESEs function as ESEs in their natural context. Like most exons, these exons contain several PESEs. Since knocking out any one of several could produce a severalfold decrease in splicing efficiency, we conclude that there is little redundancy among ESEs in an exon and that they must work in concert to optimize splicing.

Alternative Splicing↗
PubMed1997

A two-base deletion in exon 6 of the 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL) gene producing the skipping of exons 5 and 6 determines 3-hydroxy-3-methylglutaric aciduria.

A novel two-base deletion in the 3-hydroxy-3-methylglutaryl coenzyme A lyase (HL) gene was found in a Spanish patient with homozygous 3-hydroxy-3-methylglutaric aciduria. Amplification by RT-PCR of the mRNAs showed that the gene was transcribed into three different mRNAs. One showed the complete deletion of exons 5 and 6 located between nucleotides 348 and 561 of the HL cDNA. The second transcript showed deletion of exon 6 only, and the third contained a two-base deletion CT in exon 6, corresponding to nucleotides 504 and 505 of the HL cDNA. These aberrant mRNAs are predicted to encode three abnormal HMG-CoA lyase proteins; the first (from skipped exons 5 and 6) lacks 71 amino acids, which represents 24% of the mature protein; the second, (from the skipping of exon 6, producing a frameshift) contains only 192 amino acids, the last 26 of which are missense amino acids preceding a stop codon; the third contains only 175 amino acids, the last 7 of which are missense. Northern blot analysis showed that the HL mRNA levels of the patient were 4% of the control. PCR quantitative analysis indicated that the mRNA lacking exons 5 and 6 was the most abundant, representing 88% of the total. The other two mRNAs represented 8% and 4%, respectively. In the genomic DNA only one CT deletion was found at positions +7 and +8 at beginning of exon 6. No mutations were observed in the splice donor, splice acceptor, or pyrimidine-rich sequences of the intronic regions flanking exons 5 and 6. All three aberrant mRNAs resulted only from the deletion of nucleotides CT. We suggest that this deletion may affect the interaction between the small nuclear ribonucleoproteins (snRNPs) and exon 6, and that, as a result, the abnormal splicing of the pre-mRNA produces two different aberrant transcripts.

Adolescent↗
PubMed1999

A premature termination codon within an alternative exon affecting only the metabolism of transcripts that retain this exon.

Protein 4.1 pre-mRNA splicing is regulated in tissue- and development-specific manners. Exon 16, which encodes the N-terminal region of the spectrin/actin-binding domain, is one of the alternatively spliced sequence motifs. It is present in late differentiated erythroid cells but absent from early erythroblasts and from lymphoid cells. We describe a single nucleotide deletion of the erythroid protein 4.1 gene associated with hereditary elliptocytosis. The deletion located in exon 16 leads to a frameshift and a premature termination codon within the same exon. In an effort to examine the premature stop codon effect in relationship with exon 16 alternative splicing, we analyzed erythroid and lymphoid protein 4.1 mRNAs using the mutation and a linked downstream polymorphism as markers. We found that the premature stop codon does not affect the tissue-specific alternative splicing among the two cell types analyzed and that the resulting alteration of mRNA metabolism correlates with the retention of exon 16 in reticulocytes. Conversely, skipping of exon 16 in lymphoid cells converts the mutant mRNA to a normal lymphoid-specific mRNA isoform, hence bypassing the nonsense codon. Consistent with data obtained on constitutive nonsense exons, our observations argue in favor of a stop codon recognition mechanism that occurs after the regulated splicing status of the nonsense exon has been achieved.

Alternative Splicing↗
PubMed1996

Induction of exon skipping of the dystrophin transcript in lymphoblastoid cells by transfecting an antisense oligodeoxynucleotide complementary to an exon recognition sequence.

In this paper we first report that exon skipping from the dystrophin gene transcript could be induced in living cells by an antisense oligodeoxynucleotide (ODN) complementary to an exon recognition sequence (ERS). Incubation of lymphoblastoid cells with an antisense ODN against the purine-rich region of dystrophin exon 19 resulted in skipping of the exon from the dystrophin transcript. Skipping of exon 19 started to appear after 6 hours of incubation, and complete skipping was observed after 24 hours of incubation. None of the other 78 dystrophin exons were skipped, and exon 19 skipping could be induced by the sense ODN or by an antisense ODN corresponding to another ERS. These results showed that antisense ODN against ERS can induce exon skipping even in living cells and ERS is functioning as an essential cis-element for proper splicing in dystrophin pre-mRNA.

Alternative Splicing↗
PubMed1992

Partial deletion of a dystrophin gene leads to exon skipping and to loss of an intra-exon hairpin structure from the predicted mRNA precursor.

In dystrophin Kobe exon 19 of the dystrophin gene is skipped during the process of mRNA precursor splicing even though the splice sites are unchanged (Matsuo et al. J. Clin. Invest. 87:2127-2131,1991). In the predicted secondary structure of the mRNA precursor, exon 19 of dystrophin Kobe is paired with intron sequences, whereas a large part of exon sequence from wild type is paired with itself and folded into a large hairpin structure. As all of 22 additional dystrophin exons analyzed also form intra-exon hairpin structures, these structures may be considered essential components of exons. We suggest that the abolishment of a hairpin structure in the truncated exon of dystrophin Kobe might prevent the splicing machinery from recognizing the splice sites and induce exon skipping.

Base Composition↗
PubMed2002

The novel exon, exon T, of the human progesterone receptor gene and the genomic organization of the gene.

Recently, we have cloned the novel isoform of the progesterone receptor (PR) cDNA (PR isoform S cDNA) from the human testicular cDNA library. The isoform S cDNA consists of the novel exon (termed the exon S of the PR gene) and the exons 4-8 of the PR gene. In order to investigate the existence of the other isoform of the human PR cDNA, the human testicular cDNA library was screened by the exons 4-8 corresponding sequence of the human PR cDNA in the present study. As a result, we have identified a novel isoform of the PR cDNA (termed the PR isoform T cDNA (PR-T cDNA)), which consisted of a previously unidentified 5'-sequence and the exons 4-8 of the PR gene. The structure of this isoform T cDNA is essentially similar to that of the isoform S cDNA. By the genomic cloning, the 5'-sequence of the PR isoform T mRNA was demonstrated to originate from a novel independent exon, exon T, which was located in the 5'-upstream region of the exon S.

Base Sequence↗