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PubMed1997

A tripartite group II intron in mitochondria of an angiosperm plant.

In mitochondria of flowering plants the nad5 open reading frame is assembled from five exons via two conventional cis-splicing and two trans-splicing events. Trans-splicing between exons c and d in wheat, petunia and Arabidopsis involves a bipartite group II intron structure, while in Oenothera a large portion of intron domains I-IV is missing from the major genomic locus. This intron region has been lost downstream of exon c and is now found in a distant genomic region. Intragenomic recombination across an 11 nucleotide sequence has separated these intron parts, which now have to be reassembled from three independent RNA precursors. This organisation coexists with highly substoichiometric copy numbers of the bipartite intron arrangement, consistent with an evolutionary origin of the tripartite intron by genomic disruption.

Arabidopsis↗

PubMed1998

The novel function of the Saccharomyces cerevisiae CBP2 gene as a splicing factor essential to excision of the Saccharomyces douglasii LSU intron in vivo.

In the yeast Saccharomyces cerevisiae, the product of the nuclear gene CBP2 is required exclusively for the splicing of the terminal intron of the mitochondrial cytochrome b gene. The homologous gene from the related yeast, Saccharomyces douglasii, has been shown to be essential for respiratory growth in the presence of a wild-type S. douglasii mitochondrial genome and dispensable in the presence of an intronless mitochondrial genome. The two CBP2 genes are functionally interchangeable although the target intron of the S. cerevisiae CBP2 gene is absent from the S. douglasii mitochondrial genome. To determine the function of the CBP2 gene in S. douglasii mitochondrial pre-RNA processing we have constructed and analyzed interspecific hybrid strains between the nuclear genome of S. cerevisiae carrying an inactive CBP2 gene and S. douglasii mitochondrial genomes with different intron contents. We have demonstrated that inactivation of the S. cerevisiae CBP2 gene affects the maturation of the S. douglasii LSU pre-RNA, leading to a respiratory-deficient phenotype in the hybrid strains. We have shown that the CBP2 gene is essential for excision of the S. douglasii LSU intron in vivo and that the gene is dispensable when this intron is deleted or replaced by the S. cerevisiae LSU intron.

Cell Division↗

PubMed1999

An alternative intronic promoter of the Bombyx A3 cytoplasmic actin gene exhibits a high level of transcriptional activity in mammalian cells.

Previous observations have indicated that the Bombyx mori gene for A3 cytoplasmic actin and vertebrate actin genes might make use of similar mechanisms for regulation of gene expression. To examine the suggested similarities, we have analyzed the expression of a LacZ reporter plasmid construct containing the 5' and 3' regulatory regions and the first intron of the A3 actin gene in a variety of vertebrate cell lines. We found that this silkworm expression cassette could drive expression of foreign genes in both mammalian and avian cell lines. Detailed analysis, however, indicated that neither the CArG box nor any of the promoter elements previously identified in the A3 actin gene were required for expression in mammalian cells. On the other hand, the first intron contained an efficient promoter, exhibiting in mouse cells a transcriptional activity comparable to that of the SV40 early promoter. The first intron of the A3 gene was also found to contain enhancer-like DNA elements that could stimulate the heterologous SV40 early promoter in mammalian cells. Promoter activity of the first intron of the A3 actin gene has not been observed previously. Recently however, we described a rare A3 actin mRNA isoform in B. mori cells, which initiates within the first intron. We suggest that the identified intronic promoter may be active not only in vertebrate cells but also in silkworm, and that it regulates the synthesis of the alternative A3 actin mRNA isoform. The characteristics of the 5' regulatory region of the A3 gene described here can also be exploited in the construction of bi-functional insect-mammalian expression vectors.

5' Untranslated Regions↗

PubMed2005

A novel cryptic exon identified in the 3' region of intron 2 of the human dystrophin gene.

The dystrophin gene, which is mutated in Duchenne muscular dystrophy (DMD), is the largest known human gene and is characterized by the huge size of its introns. Intron 2, the second largest intron, is 170-kb long and has been shown to include a 140-bp cryptic exon (exon 2a) in its 5' region. The rest of this intron has no known function. In this study, we find that another cryptic exon, located in the 3' region of intron 2, is activated in a promoter- or tissue-specific manner. An unknown 98-bp insertion precisely between exons 2 and 3 was identified in one of the dystrophin mRNAs from lymphocytes of a DMD patient with a duplication of exon 2. This 98-bp sequence, located in the 3' region of intron 2, was found to possess a branch point, acceptor and donor splice-site consensus sequences, and an exonic splicing enhancer sequence, and thus is a novel exon, which we named "exon 2b." In lymphocytes, exon 2b incorporation was detected in the muscle-specific, promoter-driven transcript. Five of 20 normal human tissue mRNAs, including cardiac and skeletal muscle mRNAs, were confirmed to contain a fragment extending from exon 1 to exon 2b by reverse transcription PCR amplification, indicating that exon 2b is activated in a tissue-specific manner. This provides a clue to a novel cause of dystrophinopathy.

3' Untranslated Regions↗

PubMed2005

Identification of one intron loss and phylogenetic evolution of Dfak gene in the Drosophila melanogaster species group.

Intron loss and its evolutionary significance have been noted in Drosophila. The current study provides another example of intron loss within a single-copy Dfak gene in Drosophila. By using polymerase chain reaction (PCR), we amplified about 1.3 kb fragment spanning intron 5-10, located in the position of Tyr kinase (TyK) domain of Dfak gene from Drosophila melanogaster species group, and observed size difference among the amplified DNA fragments from different species. Further sequencing analysis revealed that D. melanogaster and D. simulans deleted an about 60 bp of DNA fragment relative to other 7 Drosophila species, such as D. elegans, D. ficusphila, D. biarmipes, D. takahashii, D. jambulina, D. prostipennis and D. pseudoobscura, and the deleted fragment located precisely in the position of one intron. The data suggested that intron loss might have occurred in the Dfak gene evolutionary process of D. melanogaster and D. simulans of Drosophila melanogaster species group. In addition, the constructed phylogenetic tree based on the Dfak TyK domains clearly revealed the evolutionary relationships between subgroups of Drosophila melanogaster species group, and the intron loss identified from D. melanogaster and D. simulans provides a unique diagnostic tool for taxonomic classification of the melanogaster subgroup from other group of genus Drosophila.

Amino Acid Sequence↗

PubMed1991

Inhibition of pre-mRNA splicing by antisense RNA in vitro: effect of RNA containing sequences complementary to introns.

The objective of the experiments described in this paper was to test the potential of antisense RNAs complementary to the internal portion of an intron to inhibit the splicing process and to determine the mechanism of such inhibition. The results obtained indicate that RNA fragments complementary to the internal portion of an intron can effectively inhibit the splicing of pre-mRNA. Inhibition was observed only with antisense RNA complementary to pre-mRNA suggesting that the inhibitory effect was due to the formation of a hybrid with the corresponding portion of the pre-mRNA's intron. The observed inhibition was not due to interference with possible intron elements essential for the splicing process, for the deletion of the sequences complementary to inhibitory antisense RNA from the corresponding pre-mRNA molecule did not affect the efficiency of a splicing reaction, and the addition of antisense RNA to pre-mRNA mutants carrying such deletions did not result in any inhibition. Our results indicate that the observed inhibition is a function of the length of the antisense RNA expressed as a fraction of an intron with which it interacts when antisense RNA is modified by incorporation of a "hinge" element, it loses its inhibitory potential suggesting that the inhibitory effect is probably due to limitation of conformational flexibility of an intron.

Animals↗

PubMed1992

The Euglena gracilis rbcS gene contains introns with unusual borders.

We have recently shown that, in Euglena gracilis, leader sequences are transferred by trans-splicing to the vast majority of cytoplasmic mRNAs. Trans-splicing is involved in the maturation of the rbcS transcript, which encodes eight small subunits of the ribulose 1,5 bisphosphate carboxylase/oxygenase. In this report, we show that the Euglena rbcS gene introns are different from introns found in plant rbcS genes. In addition these introns do not have the conserved 5' and 3' border sequences found in introns of eucaryotic nuclear-encoded pre-mRNAs, and they do not present any homology with self-splicing introns of groups I and II. Secondary structure analyses show that the 5' and 3' ends of Euglena introns can base-pair, suggesting that an unusual splicing mechanism exists in Euglena.

Amino Acid Sequence↗

PubMed1995

Genes encoding the beta 1 subunit of voltage-dependent Na+ channel in rat, mouse and human contain conserved introns.

We provide evidence in this study that the 86-bp insert in the beta 1.2 mRNA isoform of the voltage gated sodium channel is an intron. Transcripts still retaining this intron were detected in all tissues where the beta 1 gene expression was investigated. We also show that the exon/intron boundaries of the last two introns are conserved among rat, mouse and human beta 1 gene. Unlike the highly conserved cDNAs, introns in only the rat and mouse genes are highly related. The last intron is very short (86-90 bp) and is located in the 3' untranslated sequence, both uncommon properties of mammalian pre-mRNA introns.

Animals↗

PubMed1996

The size differences among mammalian introns are due to the accumulation of small deletions.

In order to investigate the molecular mechanisms that alter intron size, we conducted an extensive interspecies comparison of homologous introns among three mammalian groups: human, artiodactyls, and rodents. The size differences of introns were statistically significant among all three groups (longest intron was for human and shortest for rodents), and appear to be due to the accumulation of small deletions, according to the separate count of insertion and deletion frequencies. The distribution of intron size differences also has a shape similar to that for the distribution of insertion/deletion sizes found in pseudogenes. It is suggested that introns are selectively neutral to small-scale changes of the genome size, which inherently contain the bias of favoring short deletions against short insertions.

Animals↗

PubMed1996

Introns and protein revolution--an analysis of the exon/intron organisation of actin genes.

A catalogue of intron positions obtained from a large number of actin genes has been compiled with a view to understanding the possible origin of intervening sequences. Actins are ubiquitous proteins conserved in evolution and an analysis of their gene structures from various organisms has revealed that there may be at least 25 intron positions distributed at different positions in the coding regions. A comparison of intron positions from a wide range of organisms from that of yeast to human actins shows that introns could be more ancestral in origin. The conservation in the observed intron patterns within the different tissue types hints at a possible functional significance of introns in present day actin genes.

Actins↗

PubMed1988

Sequence analysis of mitochondrial DNA from Podospora anserina. Pervasiveness of a class I intron in three separate genes.

A 48 kb region of the 95 kb mitochondrial genome of Podospora anserina has been mapped and sequenced (1 kb = 10(3) base-pairs). The DNA sequence of the genes for ND2, 3, 4, ATPase 6 and URFC are presented here. As in Neurospora crassa, the ND2 and 3 genes consist of a unit separated by one TAA stop codon. ND3, 4 and ATPase 6 are interrupted by class I introns. All three introns are remarkably similar in the C-domain of their secondary structure, sufficient enough to designate them as new subgroup, class IC introns. The open reading frames of the ND3 and 4 introns bear a high sequence similarity to the open reading frame of the class IB introns of ATPase 6 from N. crassa and ND1 from Neurospora intermedia Varkud. We also show that the tRNA Met-2 gene is duplicated and is involved in a recombinational event. The 5' region of URFC is also duplicated but no involvement of this gene with recombination or formation of plasmids is known. The evolutionary significance of the similarities of intron secondary structures and open reading frames of the ND3, 4 and ATPase 6 genes is discussed, including the possible separate evolution of structural and coding sequences.

Adenosine Triphosphatases↗

PubMed1989

Protein encoded by the third intron of cytochrome b gene in Saccharomyces cerevisiae is an mRNA maturase. Analysis of mitochondrial mutants, RNA transcripts proteins and evolutionary relationships.

We have established the nucleotide sequence of the wild-type and that of a trans-acting mutant located in the third (bi3) intron of the Saccharomyces cerevisiae mitochondrial cytochrome b gene. The intron, 1691 base-pairs long, has an open reading frame 1045 base-pairs long, in phase with the preceding exon and the mutation replaces the evolutionarily conserved Gly codon of the second consensus motif by an Asp codon and blocks the formation of mature cytochrome b mRNA. Splicing intermediates of 5300 and 3900 bases with unexcised bi3 intron and a characteristic novel polypeptide (p50), the size of which corresponds to the chimeric protein encoded by upstream exons and the bi3 intronic open reading frame (ORF), accumulate in this and other bi3 splicing-deficient mutants. We conclude that the protein encoded by the bi3 ORF is a specific mRNA maturase involved in the splicing of the cytochrome b mRNA. The open reading frame of the third intron is remarkably similar to that of the unique intron of the cytochrome b gene (cob A) of Aspergillus nidulans. Both are located in exactly the same position and possibly derive from a recent common ancestor by a horizontal transfer. We have established the nucleotide sequence of an exonic mutant located in the B3 exon. This missense mutation changes the Phe codon 151 into a Cys codon and leads to the absence of functional cytochrome b but does not affect splicing. Finally, we have studied the splicing pathway leading to the synthesis of cytochrome b mRNA by analysing, in a comprehensive manner, the 22 splicing intermediates of several mutants located in bi3.

Aspergillus nidulans↗

PubMed1995

The first intron of the mouse neurofilament light gene (NF-L) increases gene expression.

Neurofilament expression is developmentally and post-transcriptionally controlled. Using transient transfection assays in mouse L cells, we demonstrate that the expression of the mouse neurofilament light subunit (NF-L) is influenced by intron sequences. NF-L expression was decreased twenty fold upon deletion of the three intron sequences. Elements contained principally within a 350 bp region of intron 1 were responsible for enhanced NF-L expression. Enhancement of expression did not occur when intron I was placed 3' to a heterologous chloramphenicol acetyl transferase (CAT) gene whose expression was driven by NF-L 5' sequences. The intron enhancement of NF-L expression was not promoter-specific and also occurred with the mouse sarcoma virus (MSV) LTR promoter. These data suggest intron sequences may be important in regulating NF gene expression.

Animals↗

PubMed1996

Group II introns: elaborate ribozymes.

Group II introns are found in organelle genomes of plants, fungi and algae as well as in some bacteria. Some group II introns have been shown to self-splice in vitro and thus constitute examples of ribozymes. Their splicing pathway is analogous to the splicing pathway of nuclear pre-mRNA introns. They thus constitute simple models to analyze RNA catalysis of this type of splicing reactions. In this review article, I will summarize our current state of understanding of the ribozyme activity of group II introns and show that their large size correlates with their ability to perform complex tasks. After discussing the similarities found between group II and nuclear pre-mRNA introns, I will briefly evoke how the ribozyme activity of group II introns might be involved in their transposition at the DNA level.

Binding Sites↗

PubMed1986

The mosaic cox1 gene in the mitochondrial genome of Schizosaccharomyces pombe: minimal structural requirements and evolution of group I introns.

The gene encoding subunit 1 of cytochrome oxidase (cox1) in the fission yeast Schizosaccharomyces pombe is polymorphic. In strain 50 it contains two group I introns with open reading frames (ORFs) in phase with the upstream exons (Lang, 1984). In strain EF1 two additional very short group I introns which do not possess ORFs were detected by DNA sequencing. These two introns (AI2a and AI3) share distinct characteristics concerning their nucleotide sequence and secondary structure and are located at identical positions as the introns AI4 and AI5 beta, respectively, in the cox1 gene of Saccharomyces cerevisiae. The sequence homology of the cob and cox1 genes around the splice points of introns AI2a, AI4, and BI4 (cob intron 4) might reflect horizontal gene transfer between the distantly related species S. pombe and S. cerevisiae.

Amino Acid Sequence↗

PubMed1988

Stability of group I intron RNA in Escherichia coli and its potential application in a novel expression vector.

Intron RNA excised from the primary transcript of the phage T4 td gene was found to be unusually stable in vivo. In contrast to the average half-life of about 1.5 min for a typical Escherichia coli mRNA at 37 degrees C, the half-life of the excised group-I td intron ranged from 12 to 19 min for the linear form and from 22 to 33 min for the circular form. A 631-nucleotide region of the intron that is not essential for splicing was replaced by the chloramphenicol acetyltransferase (CAT) structural gene (cat). Although the presence of the foreign sequence reduced intron stability several-fold, the half-life of the resulting intron-cat hybrid RNA was found to be twice that of the normal cat mRNA. The increase in stability was accompanied by a five- to eight-fold increase in CAT production above that seen with transcriptional activation from the strong Ptac promoter alone. The over-production was both temperature-dependent and partially splicing-dependent. This type of intron fusion represents a novel method of transcript stabilization, which is of potential use to augment other means of increasing gene expression for purposes of product amplification.

Chloramphenicol O-Acetyltransferase↗

PubMed1991

Two introns in the pheromone 3-encoding gene of Euplotes octocarinatus.

The portion of the pheromone 3 (Phr3)-encoding gene (phr3) of Euplotes octocarinatus, corresponding to secreted Phr3 was isolated using the polymerase chain reaction and oligodeoxyribonucleotide primers flanking the Phr3-encoding cDNA. Unexpectedly, the sequence analysis revealed that this gene is interrupted by two introns with lengths of 63 bp and 72 bp. These introns belong to the class of nuclear pre-mRNA introns and contain typical 5'- and 3'-consensus sequences, as well as unique features. Our findings constitute the first example of introns in any Euplotes species and the first case of multiple introns in hypotrichous ciliates. In ciliates such as Euplotes, the macronucleus is the transcriptionally active nucleus. It develops from a micronucleus in a process which involves chromosome breakage and the elimination of up to 95% of the micronuclear genome. The existence of the introns in the macronuclear version of phr3 shows that, in spite of this extensive elimination, some noncoding sequences are nevertheless retained in the macronucleus of hypotrichous ciliates. It was recently discovered by Meyer et al. [Proc. Natl. Acad. Sci. USA 88 (1991) 3758-3761] that the Phr3-encoding cDNA contains three in frame TGA triplets coding for cysteine. The genomic sequence of phr3 confirms this finding.

Amino Acid Sequence↗

PubMed1992

Protein-coding introns from the 23S rRNA-encoding gene form stable circles in the hyperthermophilic archaeon Pyrobaculum organotrophum.

Two archaeal introns have been discovered in the single-copy 23S rRNA-encoding gene of the hyperthermophile, Pyrobaculum organotrophum. After excision from rRNA transcripts, both introns circularize and are stably retained in the cell. Putative proteins encoded by the introns and covering most of the intron sequence share a decapeptide motif with proteins encoded by another archaeal intron and by group I introns.

Amino Acid Sequence↗