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PubMed2003

Cis-acting intronic elements that regulate cartilage-specific alternative splicing of the type II collagen (Col2) pre-mRNA lie at or near splice site junction sequences flanking exon 2 of the gene.

UNLABELLED: Knowledge of the cis-acting elements is required for identifying trans-acting splicing factors underlying cartilage-specific alternative splicing of Col2 pre-mRNA. By performing desired deletions in the mouse Col2 pre-mRNA, location of the intronic cis-acting elements was narrowed down to be at or near splice-junction sequences flanking exon 2 of the gene. INTRODUCTION: Type II collagen (Col2) pre-mRNA undergoes cartilage-specific alternative splicing involving exon 2 during chondrocyte differentiation. Thus, the trans-acting protein factors that regulate the splicing are associated with the differentiation of chondrocytes. Knowledge of the cognate cis-acting elements is necessary to eventually identify the trans-acting factors. MATERIALS AND METHODS: To localize the cis-acting sequences, we created several deletions within a minigene containing exon 1 to exon 4 of mouse Col 2 gene and evaluated alternative splicing of the resulting pre-mRNAs in ATDC5 cells, a model of insulin-stimulated chondrocyte differentiation. The first deletion reduced intron 1 from 3799 to 259 bp, the second reduced intron 2 from 1108 to 94 bp, the third combined the above two deletions, and the fourth was derived from the third by removing intron 3 and exon 4. ATDC5 cells harboring these constructs were cultured for up to 21 days with or without insulin. Alternative splicing was evaluated by determining the ratio of Col2B (lacks exon 2) to Col2A (has exon 2) RNAs by reverse transcription-polymerase chain reaction. RESULTS: The deletion in intron 1 had no effect on the alternative splicing while other deletions affected splicing (demonstrated by the presence of splicing intermediates) in cells cultured without insulin or with insulin for 1 week. The splicing intermediates were not seen from any construct when cells were cultured longer (14-21 days) with insulin. CONCLUSION: These results show that the 259-bp intron 1, the 94-bp intron 2, and exon 2 sequences retained in the fourth construct provide cis-acting signal sufficient for insulin-induced cartilage-specific alternative splicing of Col2 pre-mRNA.

Alternative Splicing↗

PubMed2006

Intronic binding sites for hnRNP A/B and hnRNP F/H proteins stimulate pre-mRNA splicing.

hnRNP A/B proteins modulate the alternative splicing of several mammalian and viral pre-mRNAs, and are typically viewed as proteins that enforce the activity of splicing silencers. Here we show that intronic hnRNP A/B-binding sites (ABS) can stimulate the in vitro splicing of pre-mRNAs containing artificially enlarged introns. Stimulation of in vitro splicing could also be obtained by providing intronic ABS in trans through the use of antisense oligonucleotides containing a non-hybridizing ABS-carrying tail. ABS-tailed oligonucleotides also improved the in vivo inclusion of an alternative exon flanked by an enlarged intron. Notably, binding sites for hnRNP F/H proteins (FBS) replicate the activity of ABS by improving the splicing of an enlarged intron and by modulating 5' splice-site selection. One hypothesis formulated to explain these effects is that bound hnRNP proteins self-interact to bring in closer proximity the external pair of splice sites. Consistent with this model, positioning FBS or ABS at both ends of an intron was required to stimulate splicing of some pre-mRNAs. In addition, a computational analysis of the configuration of putative FBS and ABS located at the ends of introns supports the view that these motifs have evolved to support cooperative interactions. Our results document a positive role for the hnRNP A/B and hnRNP F/H proteins in generic splicing, and suggest that these proteins may modulate the conformation of mammalian pre-mRNAs.

Base Sequence↗

PubMed2005

Gametophytic selection in Arabidopsis thaliana supports the selective model of intron length reduction.

Why do highly expressed genes have small introns? This is an important issue, not least because it provides a testing ground to compare selectionist and neutralist models of genome evolution. Some argue that small introns are selectively favoured to reduce the costs of transcription. Alternatively, large introns might permit complex regulation, not needed for highly expressed genes. This "genome design" hypothesis evokes a regionalized model of control of expression and hence can explain why intron size covaries with intergene distance, a feature also consistent with the hypothesis that highly expressed genes cluster in genomic regions with high deletion rates. As some genes are expressed in the haploid stage and hence subject to especially strong purifying selection, the evolution of genes in Arabidopsis provides a novel testing ground to discriminate between these possibilities. Importantly, controlling for expression level, genes that are expressed in pollen have shorter introns than genes that are expressed in the sporophyte. That genes flanking pollen-expressed genes have average-sized introns and intergene distances argues against regional mutational biases and genomic design. These observations thus support the view that selection for efficiency contributes to the reduction in intron length and provide the first report of a molecular signature of strong gametophytic selection.

Arabidopsis↗

PubMed2006

Introns regulate RNA and protein abundance in yeast.

The purpose of introns in the architecturally simple genome of Saccharomyces cerevisiae is not well understood. To assay the functional relevance of introns, a series of computational analyses and several detailed deletion studies were completed on the intronic genes of S. cerevisiae. Mining existing data from genomewide studies on yeast revealed that intron-containing genes produce more RNA and more protein and are more likely to be haplo-insufficient than nonintronic genes. These observations for all intronic genes held true for distinct subsets of genes including ribosomal, nonribosomal, duplicated, and nonduplicated. Corroborating the result of computational analyses, deletion of introns from three essential genes decreased cellular RNA levels and caused measurable growth defects. These data provide evidence that introns improve transcriptional and translational yield and are required for competitive growth of yeast.

Actins↗

PubMed2005

C/EBPalpha regulates human adiponectin gene transcription through an intronic enhancer.

Adiponectin is an adipose-derived hormone that enhances insulin sensitivity and plays an important role in regulating energy homeostasis. Here, we demonstrate that the DNA encoding the first intron of the human adiponectin gene contains an intronic enhancer that regulates adiponectin gene expression in an adipose tissue-specific manner. Insertion of the DNA encoding the first intron into reporter constructs containing the proximal adiponectin promoter (Pro-Int1-Luc) resulted in a 20-fold increase in activity relative to the promoter alone in 3T3-L1 adipocytes. Coexpression of CCAAT/enhancer-binding protein (C/EBP)alpha increased luciferase activity of the Pro-Int1-Luc construct approximately 75-fold but had no effect on the constructs containing the proximal adiponectin promoter alone. At least eight potential C/EBPalpha response elements are located between +3000 to +10000 nucleotides within the DNA encoding the first intron, including a 34-bp core sequence for the intronic enhancer that contains three tandem C/EBPalpha response elements. However, the intronic enhancer is not conserved between human and mouse. Overexpression or siRNA-mediated knockdown of endogenous C/EBPalpha significantly increased or decreased, respectively, adiponectin mRNA levels in differentiated human Chub-S7 adipocytes, while neither C/EBPbeta nor C/EBPdelta significantly affected adiponectin expression in mature adipocytes. Thus, C/EBPalpha is a key transcription factor for full activation of human adiponectin gene transcription in mature adipocytes through interaction with response elements in the intronic enhancer.

Adipocytes↗

PubMedDate not supplied

Allelic variation in the intron 2 and 3 of the MICA gene.

We have determined by sequencing the allelic variation in intron 2 and 3 of hte MICA gene for a total of 22 different alleles. Sequencing of introns was performed in two directions, using DNA from homozygous cell lines from families and from unrelated individuals. Intron 2 is 273 bp long and did in the alleles investigated not reveal any length polymorphism. We found a total of eight polymorphic positions which exhibit a strict biallelism, as it is also found in the polymorphisms for exon 2, 3 and 4 of MICA. Intron 3 is 586 bp long an required an additional set of primers placed near the middle of this intron in order to allow a complete bidirectional sequence. In intron 3, a total of 10 polymorphic positions were identified. Interestingly, we found two variants of the allele MICA*002 which are distinguished only by one basepair difference in intron 3. The variant MICA*002A is associated with HLA-B35 and B58, while the allele MICA*002B is associated with B38 and B39.

Alleles↗

PubMed2001

Organization and regulation of the rat Cx31 gene. Implication for a crucial role of the intron region.

The connexin31 (Cx31) gene, a member of the connexin multigene family, is expressed in a characteristic spatiotemporal pattern during placental development in rodents. To elucidate the trophoblast-specific regulation of Cx31, we have isolated the rat Cx31 gene and performed structural and functional promoter analysis. The isolated Cx31 gene contains two exons separated by an intron of 2.6 kb. The first exon of the Cx31 gene is preceded by a TATA-less promoter region. Transcription is initiated in exon 1 from two transcription start sites producting transcripts of 105 and 139 bp. The 935 bp of the 5' flanking region of exon 1 comprises five putative binding sites for the GATA transcription factors as well as a NF-kappa B element, a CAAT-box and E-box/E-box-related sequences. For functional promoter analysis, the rat choriocarcinoma cell line Rcho-1 and the mouse keratinocyte cell line Hel37, which both express Cx31, were chosen. Only constructs including exon 1 and the complete intron showed high activity in transient transfection experiments in both cell lines. All deletion fragments of the putative promoter region, but which contain the entire intron sequence, did not reveal any obvious changes in luciferase activity. However, deletion of 1.1 kb of the intron sequence downstream of the splice donor site resulted in the loss of promoter activity. The intron exhibits no enhancer activity for the gene; however, the mRNA stability was increased in the presence of the intron sequence. These results indicate that parts of the intron sequence are critical for basic promoter function of the Cx31 gene.

Animals↗

PubMed2001

A factor VIII minigene comprising the truncated intron I of factor IX highly improves the in vitro production of factor VIII.

The biosynthesis of coagulation factor VIII (FVIII) is hampered by successive controls that limit its production. To improve this production, a truncated intron I sequence of factor IX (TFIXI1) was inserted in FVIII cDNA in place of FVIII introns 1, 12 and 13 and also as a combination between introns 1 and 12, and introns 1 and 13. The intron 12 and 13 locations were targeted because this region was previously shown to contain a transcriptional silencer. The expression of FVIII in CHO and HepG2 cells revealed important variations in the properties of the minigenes depending on the TFIXI1 insertion sites. In FVIII intron 13 location the TFIXI1 seemed to diminish the transcriptional silencer activity, whereas it was poorly spliced in intron 12 position. Among the five constructs, FVIII I1+13 leaded to a significant improvement in FVIII secretion (13 times) that was associated with a dramatic intracellular accumulation in cells. Therefore, the FVIII I1+13 minigene could represent a particular interest to produce recombinant FVIII in vitro as well as in the aim of gene therapy of haemophilia A.

Animals↗

PubMed1992

Two homologous mitochondrial introns from closely related Saccharomyces species differ by only a few amino acid replacements in their Open Reading Frames: one is mobile, the other is not.

We have undertaken a comprehensive study of the gene conversion of all the mitochondrial introns of Saccharomyces capensis. The approach used involved the measurements of intron transmission amongst the progeny of crosses between a recipient strain (Saccharomyces cerevisiae intronless mitochondria) and various donor strains (Saccharomyces capensis, with various combinations of mitochondrial introns). We have shown that the S. capensis second intron (bi2 of cytochrome b gene) is extremely active as a donor in gene conversion whereas its homologous S. cerevisiae intron is not. Determination of sequence of the S. capensis intron demonstrates that it differs from that of the homologous S. cerevisiae intron (bi2) by a very small number of nucleotide substitutions.

Amino Acid Sequence↗

PubMed1992

The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo.

RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several proteins encoded by nuclear genes. In this study we report on the isolation, nucleotide sequence, and possible functions of the nuclear MRS2 gene. When present on high copy number plasmids, the MRS2 gene acts as a suppressor of various mitochondrial intron mutations, suggesting that the MRS2 protein functions as a splicing factor. This notion is supported by the observations that disruption of the single chromosomal copy of the MRS2 gene causes (i) a pet- phenotype and (ii) a block in mitochondrial RNA splicing of all four mitochondrial group II introns, some of which are efficiently self-splicing in vitro. In contrast, the five group I introns monitored here are excised from pre-mRNA in a MRS2-disrupted background although at reduced rates. So far the MRS2 gene product is unique in that it is essential for splicing of all four group II introns, but relatively unimportant for splicing of group I introns. In strains devoid of any mitochondrial introns the MRS2 gene disruption still causes a pet- phenotype and cytochrome deficiency, although the standard pattern of mitochondrial translation products is produced. Therefore, apart from RNA splicing, the absence of the MRS2 protein may disturb the assembly of mitochondrial membrane complexes.

Amino Acid Sequence↗

PubMed2004

[Cloning and sequencing of the introns of UCP gene and construction of molecular phylogenetic tree in chicken].

The UCP genes were the newly discovered genes that can increase the energy expenditure and involve in the metabolism of fat and regulation of energy. Four pairs of primers in chicken UCP exon region were designed to amplify the introns of chicken UCP gene according to the splice ways of the mouse UCP2 gene (Accession No.AF096288). The sequence results showed that the chicken UCP gene also had five GT-AG type introns. The molecular phylogenetic tree was constructed based on the sequence of cds, intron 2 and intron 3 region, respectively. The phylogenetic tree based on the UCP cds region was consistent with the species phylogenetic tree. This result implicated that UCP gene can be regarded as the useful gene for the study of animal phylogenesis. On the contrast, the phylogenetic tree based on the intron 2 and intron 3 region was different from the species phylogenetic tree, which showed that the evolution of intron and cds region is different.

Animals↗

PubMed1990

Identical G+1 to A mutations in three different introns of the type III procollagen gene (COL3A1) produce different patterns of RNA splicing in three variants of Ehlers-Danlos syndrome. IV. An explanation for exon skipping some mutations and not others.

Identical G+1 mutations in three different introns of the gene for type III procollagen (COL3A1) that cause aberrant splicing of RNA were found in three probands with life-threatening variants of Ehlers-Danlos syndrome. Because the three mutations were in a gene with multiple and homologous exons, they provided an interesting test for factors that influence aberrant splicing. The G+1 to A mutation in intron 16 caused extensive exon skipping, the G+1 to A mutation in intron 20 caused both use of a cryptic splice site and retention of all the intron sequences, and the G+1 to A mutation in intron 42 caused efficient use of a single cryptic splice site. The different patterns of RNA splicing were not explained by evaluation of potential cryptic splice sites in the introns by either their homology with 5'-splice sites from other genes or by their delta G(0)37 values for binding to U1 RNA. Instead, the results suggested that the patterns of aberrant RNA splicing were primarily determined by the relative rates at which adjacent introns were normally spliced.

Adenine↗

PubMed1990

The discovery of new intron-containing human tRNA genes using the polymerase chain reaction.

Introns in transfer RNA genes are rare in vertebrates. Until now, the only intron-containing human tRNA genes were believed to be those coding for tRNA(Tyr). All of these introns are inserted 3' to the anticodon position in these genes. We have designed polymerase chain reaction primers that can amplify all of the tRNA(Tyr) genes for cloning and sequencing by using the conserved portions of the gene coding for the structural part of the tRNA. Our preliminary results have revealed five tRNA(Tyr) genes, each of which contains a different intron. We used the same technique to amplify, clone, and sequence the human genes for tRNA(Leu)CAA. This has resulted in the discovery that this human tRNA gene family also has introns inserted 3' to the anticodon. This polymerase chain reaction technique is useful in detecting new families of intron-containing tRNA genes as well as identifying sequence variations in the introns of individual genes.

Base Sequence↗

PubMed1989

Altered myc gene transcription and intron-induced stabilization of myc RNAs in two mouse plasmacytomas.

Accumulation of unusually high amounts of larger-than-normal c-myc mRNAs occurs in two mouse plasmacytomas, TEPC 1165 and TEPC 2027. Southern blot and DNA sequence analyses showed that both tumors have undergone translocations of immunoglobulin heavy chain loci to positions 5' of the c-myc gene promotors resulting in removal of DNA sequences encoding a negative transcriptional regulatory element. In contrast to other mouse plasmacytomas, TEPC 1165 and TEPC 2027 rearranged myc genes show increased transcription, partially explaining their abundance of myc RNA. Similar to other mouse plasmacytomas, the abundance of myc RNA in TEPC 1165 and TEPC 2027 is also influenced by increased stability of structurally atypical myc RNAs. Two myc mRNAs are found in TEPC 2027, a 2.4 kb species including all 3 myc exons and a 4.0 kb species with the 3 exons plus the first intron. The two major myc mRNAs in TEPC 1165, 3.0 and 3.9 kb species, also include all three myc exons plus portions of the first intron. S1 nuclease protection analyses show that the 5' initiation and 3' untranslated (UT) regions of the unusual TEPC 1165 RNAs are normal showing that the size differences arise solely from inclusion of first intron sequences in the large myc RNAs. DNA sequence analysis showed that the presence of first intron sequences in the large myc RNAs is due to mutations affecting the splice donor region at the 3' end of exon 1 in both tumors. SDS-PAGE analysis of immunoprecipitated TEPC 1165 and TEPC 2027 myc proteins showed them to be of normal electrophoretic mobility but no more abundant than in a pre-B cell line 18-81 that contains at least 10 fold less myc RNA. The 4.0 kb myc mRNA of TEPC 2027 is atypically stable while the 2.4 kb myc mRNA undergoes normal rapid turnover within the same cell, demonstrating that the presence of first intron sequences in the large myc RNA stabilizes it despite the presence of 3' UT and putative exon 1 destabilizing sequences. These results show that myc intron 1 sequences can counteract the effect of 3' UT region destabilizing sequences in myc RNA and suggest that the increased myc RNA stability noted in TEPC 1165 and TEPC 2027 is largely due to the presence of the intron 1 sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1988

A tRNA(Trp) intron endonuclease from Halobacterium volcanii. Unique substrate recognition properties.

We report on the properties of a partially purified tRNA intron endonuclease from the archaebacterium Halobacterium volcanii. This enzyme is capable of precise excision of the 104-nucleotide intron from halo-bacterial pre-tRNA(Trp) substrates generated in vitro by T7 RNA polymerase transcription. The reaction requires divalent cations (Mg2+ or Ca2+) or spermidine, is inhibited by monovalent cations, and produces 5'-hydroxyl and 2',3'-cyclic phosphate termini. Unlike the universal substrate recognition properties characteristic of the eukaryotic tRNA intron endonucleases, this enzyme is specific for halophilic tRNA(Trp) substrates. The partially purified enzyme is not capable of removing the intron from a yeast pre-tRNA(Phe) substrate. Analysis of the enzyme's ability to cleave tRNA(Trp) substrates lacking exon sequences demonstrated that the mature tRNA-like structure is not required in the substrate. A substrate retaining the intact intron and only the anticodon stem and loop exon regions was efficiently cleaved. Deletions within the intron indicated that the intron was not a primary site for recognition by the endonuclease; however, its presence affects the efficiency of the cleavage reaction. The possible relationship of this enzyme to other RNA endonucleases is discussed.

Base Sequence↗

PubMed1994

Multiple group II self-splicing introns in mobile DNA from Escherichia coli.

By PCR (polymerase chain reaction) amplification and cloning, we have identified four group II self-splicing introns encoding proteins related to reverse transcriptases in natural Escherichia coli isolates belonging to the ECOR collection. One intron, IntD, interrupts a DNA sequence virtually identical to that of the previously described IS3411 Insertion Sequence. A second intron, IntC, is located within an open reading frame that is closely related to a reading frame in the T-DNA of Agrobacterium tumefaciens. Finally, introns IntA and IntB are inserted at two distinct sites in one of the Rhs elements of E. coli. A comparison of their open reading frames shows that the two Rhs introns are more closely related to each other than to any other known group II intron: this suggests that transposition of group II introns may occur preferentially in cis, along the same piece of DNA.

DNA Transposable Elements↗

PubMedDate not supplied

[A model of positive rRNA gene expression regulation based on complementary interaction of transcripts of the prealbumin gene introns with its promotors].

The present paper is concerned with computation of the homology between the promoter and spacer sequences of the rRNA gene and the prealbumin introns. Both the sense and antisense strands of the promoters were compared with the introns. The rRNA promoters were found to contain a considerable number of prealbumin-homologous intron sites on the coding strand only, whereas the spacer sequence had the intron-homologous sites on both strands. The fragments homologous to the ribosome gene promoter regions are mainly concentrated in the first prealbumin intron. The oncogene, glycoprotein and the interleukin intron fragments revealed no sites homologous to the ribosome gene promoter. A model has been proposed for positive regulation of the rRNA gene expression via complementary interaction between the transcripts of the prealbumin gene introns with sense and antisense strands of the rRNA promoters. It is believed that either positive or negative regulation of the ribosome biogenesis can be achieved in this way.

Base Sequence↗

PubMed1998

Pre-mRNA processing enhancer (PPE) element increases the expression of an intronless thymidylate synthase gene but does not affect intron-dependent S phase regulation.

The pre-mRNA processing enhancer (PPE) element is an RNA sequence element derived from the intronless HSV-TK gene. Insertion of the element into the highly intron-dependent human beta-globin gene leads to efficient expression in the absence of splicing. We have analyzed the effect of the PPE element on the expression of mouse thymidylate synthase (TS) minigenes. We have previously shown that the expression of intronless TS minigenes is moderately (up to 20-fold) stimulated by the inclusion of introns. Furthermore, S phase-specific expression of TS minigenes in growth-stimulated cells depends on the presence of a spliceable intron as well as the TS promoter. The goal of our study was to determine if the PPE element would overcome the dependence on introns for efficient expression and for S phase-specific expression of transfected TS minigenes. We found that insertion of the PPE element into an intronless TS minigene partially overcame intron dependence. However, the increase in expression was much less than that observed for the intronless beta-globin gene. We also found that intronless TS or HSV-TK genes that contained the PPE element and that were driven by the TS promoter were expressed at a constant level in serum-stimulated cells. However, when an intron was included in these genes, they were expressed in an S phase-specific manner. Thus the PPE element was not able to overcome the dependence on introns for S phase-specific expression of TS minigenes.

Animals↗