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PubMed1993

Small subunit rDNA variation in a population of lichen fungi due to optional group-I introns.

A natural population of the lichen-forming ascomycetous fungus, Cladonia chlorophaea, contained individuals with small subunit ribosomal DNA (SSU rDNA) of at least four different size classes and nine restriction-site patterns. The source of these differences was the variable occurrence of 200-400-nucleotide insertions, previously identified as small group-I introns, at five different positions within the SSU coding region. By specific amplification of the sequences flanking these five intron positions with the polymerase chain reaction (PCR), a minimum of nine types of rDNA repeats were defined that differ in number, position, restriction pattern and size of introns. The positions of the introns were verified by sequence analysis. The variable distribution of these introns suggests that they are currently mobile--either by intron insertion, deletion or both--within this species complex.

Ascomycota↗

PubMed1994

A modified group I intron can function as both a ribozyme and a 5' exon in a trans-exon ligation reaction.

Here, we show that a single RNA molecule derived from a group-I intron can provide the catalytic activity, the substrate recognition domain and the attacking nucleophile in a reaction that mimics the exon ligation step of splicing. To accomplish this reaction, we have linked a 5' exon sequence to the 3' end of an attenuated form of the self-splicing Tetrahymena rRNA intron. The ribozyme (I-E1) attacks an oligoribonucleotide analog of the 3' splice site (I'-E2) to generate a product containing ligated exons (I-E1-E2) and a small intron fragment (I'). Two modified introns were constructed and tested for activity. A construct designed to interact with the 3' splice site through intermolecular P9.0 and P10 helices was found to be inactive due to failure to form a stable ribozyme-substrate complex. A second modified intron and substrate combination was engineered, in which the complex was further stabilized by an intermolecular P9.2 helix. In this case, stable complexes and reaction products were identified. The reaction efficiency was low compared to splicing of the unmodified intron-containing precursor, and will be optimized in future experiments. Following optimization, we believe that this system may be exploited to examine the functional consequences of a wide variety of 3' splice-site modifications, and may provide the basis for development of highly selective trans-acting ribozymes.

Animals↗

PubMed1994

The effect of intron sequences on expression levels of Ig cDNAs.

Several cDNA expression vectors were constructed and tested by stable transfection into a murine lymphoid cell line in order to compare secretion rates of a human immunoglobulin (Ig) light chain (LC). When the cDNA was under transcriptional control of the SV40 promoter and enhancer and preceded by the SV40 19S late mRNA intron, a weak LC production was detected. Secretion rate was not improved by replacing the SV40 promoter and enhancer by a combination of a murine Ig heavy chain (HC) gene promoter and enhancer even with insertion of additional Ig enhancers. In contrast, replacement of the 19S intron by a large intron derived from a human Ig HC gene and containing the intronic enhancer dramatically increased the secretion rate. High-level production was also obtained with the same enhancer-containing intron placed downstream from the LC cDNA. Stable transfectants were obtained that secreted the human LC in amounts comparable to those obtained with Ig genes. Our results suggest that the SV40 19S late mRNA intron used in several expression vectors is not appropriate when the purpose is to produce large amounts of antibody molecules. By providing transcriptional, splicing and polyadenylation signals, the presently described vectors will be useful for easy cloning and high-level expression in lymphoid cells of cDNAs or PCR products encoding antibody molecules.

Animals↗

PubMed1993

Translational introns: an additional regulatory element in gene expression.

The linear expression of a gene can be interrupted by the well-known RNA introns and the recently discovered protein introns. In both cases, splicing mechanisms physically excise the unexpressed segments. In this article we describe a third category of introns that we call 'translational introns'. These functional introns are not excised through a splicing mechanism; instead, the translational machinery bypasses a segment of the coding sequence of an mRNA. We suggest that 'translational introns' are part of a regulatory mechanism that may sense changes in the rate of translation and thereby control the ratio of alternative gene products.

Base Sequence↗

PubMed2007

Relationship between mRNA stability and intron presence.

Introns were found to enhance almost every steps of gene expression except increasing mRNA stability. By analyzing the genome-wide data of mRNA stability published by someone previously, we found that human intron-containing genes have more stable mRNAs than intronless genes, and the Arabidopsis thaliana genes with the most unstable mRNAs have fewer introns than other genes in the genome. After controlling for mRNA length, we found mRNA stability is still positively correlated with intron number in human intron-containing genes. But in yeast Saccharomyces cerevisiae, two different datasets on mRNA half-life gave conflicting results. The components of messenger ribonucleoprotein particles recruited during intron splicing may be retained in cytoplasmic mRNPs and act as signals of mRNA stability or simply insulators to avoid mRNA degradation.

Arabidopsis↗

PubMed2005

Influence of the 5' intron in the control of acetylcholinesterase gene expression during myogenesis.

During myogenesis, marked increases in both acetylcholinesterase (AChE) and its encoding mRNA are observed. The intron in the AChE gene between non-coding exon 1 [T.L. Rachinsky, S. Camp, Y. Li, T.J. Ekstrom, M. Newton, P. Taylor, Molecular cloning of mouse acetylcholinesterase: tissue distribution of alternatively spliced mRNA species, Neuron 5 (1990) 317-327] and start-site containing exon 2 [A. Mutero, S. Camp, P. Taylor, Promoter elements of the mouse acetylcholinesterase gene, J. Biol. Chem. 270 (4) (1995) 1866-1872] appears to be responsible for the enhanced expression of the enzyme upon myoblast to myotube differentiation. Deletion of a 255 bp sequence within the first intron of the AChE gene abolishes the increase in cell-associated activity observed with differentiation. To study the involvement of the intronic region in post-transcriptional processing of AChE message, we used real time RT-PCR to quantify spliced and unspliced message levels in myoblasts and myotubes. We observe a 200-fold increase of the fully spliced mRNA associated with myotube formation, while the increase in the unspliced mRNA retaining either intron 1 or intron 2 is only 5 to 15-fold. We have generated knockout mice without the conserved region of intron 1. The mice show a phenotype where skeletal muscle, hematopoietic and central nervous system AChE expression differ with the greatest effect existing in the disappearance of skeletal muscle expression [S. Camp, L Zhang, M. Marquez, B. de La Torre, J.M. Long, G. Bucht, P. Taylor, Acetylcholinesterase (AChE) gene modification in transgenic animals: functional consequences of selected exon and regulatory region deletion, VIII IMC Proceedings].

Acetylcholinesterase↗

PubMed2004

Sequence variability in the fibroin-H intron of domesticated and wild silk moths.

The single intron of the heavy-chain fibroin gene in domesticated (Bombyx mori) and wild (B. mandarina) silk moths has a length of approximately 1000 nucleotides. It is located only 57 bp from the gene's core promoter and harbors multiple AT-rich regulatory elements that have been found to enhance the basal level of transcription in vitro. In this work, the intronic nucleotide variability among members of both Bombyx species is analyzed. The intron sequences of B. mori strains k120 and Nistari, as well as B. mandarina specimens from Japan and Korea, were obtained. This information was compared with the previously reported sequences of B. mori strains p50 and C-108, as well as an additional B. mandarina specimen collected in Japan. We found a total of 26 variant positions, including variants shared by members of both species and species-specific changes. The potential functional role of these variants was investigated by using the program MatInspector to search for putative binding sites of transcription factors within the intron. We detected a multitude of putative binding elements distributed along the entire intronic sequence. Among them, 22 correspond to protein binding domains that are known to regulate fibroin transcription. The mapping of multiple variant positions within these putative binding sequences as well as in known regulatory elements of the intron argue for functional significance on the regulation of transcription.

Animals↗

PubMed2004

An intronic mutation causes long QT syndrome.

OBJECTIVES: The purpose of this research was to determine whether an intronic variant (T1945+6C) in KCNH2 is a disease-causing mutation, and if expanded phenotyping criteria produce improved identification of long QT syndrome (LQTS) patients. BACKGROUND: Long QT syndrome is usually caused by mutations in conserved coding regions or invariant splice sites, yet no mutation is found in 30% to 50% of families. In one such family, we identified an intronic variant in KCNH2. Long QT syndrome diagnosis is hindered by reduced penetrance, as the long QT phenotype is absent on baseline electrocardiogram (ECG) in about 30%. METHODS: Fifty-two family members were phenotyped by baseline QTc, QTc maximum on serial ECGs (Ser QTc-max), and on exercise ECGs (Ex QTc-max) and by T-wave patterns. Linkage analysis tested association of the intronic change with phenotype. The consequences of T1945+6C on splicing was studied using a minigene system and on function by heterologous expression. RESULTS: Expanded phenotype/pedigree criteria identified 23 affected and 29 unaffected. Affected versus unaffected had baseline QTc 484 +/- 48 ms versus 422 +/- 20 ms, Ser QTc-max 508 +/- 48 ms versus 448 +/- 10 ms, Ex QTc-max 513 +/- 54 ms versus 444 +/- 11 ms, and LQT2 T waves in 87% versus 0%. Linkage analysis demonstrated a logarithm of odds score of 10.22. Splicing assay showed T1945+6C caused downstream intron retention. Complementary deoxyribonucleic acid with retained intron 7 failed to produce functional channels. CONCLUSIONS: T1945+6C is a disease-causing mutation. It alters KCNH2 splicing and cosegregates with the LQT2 phenotype. Expanded ECG criteria plus pedigree analysis provided accurate clinical diagnosis of all carriers including those with reduced penetrance. Intronic mutations may be responsible for LQTS in some families with otherwise negative mutation screening.

Adult↗

PubMed2006

The role of introns in repeat protein gene formation.

Genes composed of tandem repetitive sequence motifs are abundant in nature and are enriched in eukaryotes. To investigate repeat protein gene formation mechanisms, we have conducted a large-scale analysis of their introns and exons. We find that a wide variety of repeat motifs exhibit a striking conservation of intron position and phase, and are composed of exons that encode one or two complete repeats. These results suggest a simple model of repeat protein gene formation from local duplications. This model is corroborated by amino acid sequence similarity patterns among neighboring repeats from various repeat protein genes. The distribution of one- and two-repeat exons indicates that intron-facilitated repeat motif duplication, in which the start and end points of duplication are located in consecutive intronic regions, significantly exceeds intron-independent duplication. These results suggest that introns have contributed to the greater abundance of repeat protein genes in eukaryotic versus prokaryotic organisms, a conclusion that is supported by taxonomic analysis.

Animals↗

PubMed2006

RNA splicing: group I intron crystal structures reveal the basis of splice site selection and metal ion catalysis.

The group I intron has served as a model for RNA catalysis since its discovery 25 years ago. Four recently determined high-resolution crystal structures complement extensive biochemical studies on this system. Structures of the Azoarcus, Tetrahymena and bacteriophage Twort group I introns mimic different states of the splicing or ribozyme reaction pathway and provide information on splice site selection and metal ion catalysis. The 5'-splice site is selected by formation of a conserved G.U wobble pair between the 5'-exon terminus and the intron. The 3'-splice site is identified through stacking of three base triples, in which the middle triple contains the conserved terminal nucleotide of the intron, OmegaG. The structures support a two-metal-ion mechanism for group I intron splicing that might have corollaries to group II intron and pre-mRNA splicing by the spliceosome.

Binding Sites↗

PubMed2005

Influence of intron and exon splicing enhancers on mammalian cell expression of a truncated spike protein of SARS-CoV and its implication for subunit vaccine development.

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) is important for vaccine development. A truncated S protein of the TW1 strain, STR2 (88 kDa), carrying three S fragments (S74-253, S294-739, and S1129-1255) was investigated to study the influences of intron and exon splicing enhancers to improve STR2 protein expression in mammalian cells. Our results showed that STR2 protein expression with the use of an 138 base-pair intron addition increased by 1.9-, 2.5-, and 4.1-fold in Vero E6, QBI-293A cells, and CHO/dhFr- cells (dihydrofolate reductase [dhfr] gene deficient CHO cells), respectively. Using the exon splicing enhancers, including a bidirectional splicing enhancer (BSE) or an exon splicing enhancer derived from the EDA alternative exon of the fibronectin gene (EDA ESE), were also found to increase STR2 protein expression in CHO/dhFr- cells by 1.7- and 2.6-fold. Nevertheless, combination of the intron and the exon splicing enhancers resulted in suppressing the intron-enhancing e STR2 protein expression in in CHO/dhFr- cells. Our studies also demonstrated the STR2 protein was mainly as the Endo H-sensitive glycoprotein (115 kDa) expressed in Vero E6, QBI-293A, and CHO/dhFr- cells. However, only a minor form of the Endo H-resistant glycoproteins ( approximately 130 kDa) was detected in CHO/dhFr- cells. Taken together, our results indicated that intron had a better enhancing effect on STR2 protein expression than exon splicing enhancers, and the expression of approximately 130 kDa STR2 glycoprotein was enhanced by the intron addition into the expression vector construct. Results of the present study can provide an optimal strategy to enhance SARS-CoV S protein expression in mammalian cells and may contribute to the development of SARS-CoV subunit vaccine.

Animals↗

PubMed2005

Complete, precise, and innocuous loss of multiple introns in the currently intronless, active cathepsin L-like genes, and inference from this event.

Retrotransposition typically generates pseudogenes. Here we demonstrate a different fate of the retro-processed genes through a novel mechanism in which the retro-processed genes still maintain their sequence intactness and the original functions. We show that the shrimp cathepsin L (CatL) gene MeCatL has lost all of its five introns. Also, ProEPB, the ancestor of the CatL-like barley EPBs and rice REP1, has lost all of its three introns. The multiple introns in a gene might have been eliminated simultaneously and precisely at the original locus for the CatL-like genes of shrimp, barley, rice, Drosophila, and Theileria. We reason that retrotransposition is not responsible for the generation of a processed active intronless (PAI) gene when the gene product retains its sequence intactness and its original function. We propose that double-strand-break repair (DSBR) machinery might play a role in cDNA-mediated homologous recombination (cDMHR) that causes the loss of introns. The cDMHR/DSBR pathway is probably a fundamental mechanism for intron loss in PAI genes and in some asymmetric-intron genes.

Amino Acid Sequence↗

PubMed1997

Characterization of nuclear tRNA(Tyr) introns: their evolution from red algae to higher plants.

We have previously isolated numerous intron-containing nuclear tRNA(Tyr) genes derived from either monocotyledonous (Triticum) or dicotyledonous (Arabidopsis, Nicotiana) plants by screening the corresponding genomic phage libraries with a synthetic tRNA(Tyr)-specific oligonucleotide. Here we have characterized additional tRNA(Tyr) genes from phylogenetically divergent plant species representing red algae (Champia), brown algae (Cystophyllum), green algae (Ulva), stonewort (Chara), liverwort (Marchantia), moss (Polytrichum), fern (Rumohra) and gymnosperms (Ginkgo) using amplification of the coding sequences from the corresponding genomic DNAs by polymerase chain reaction (PCR). All novel tRNA(Tyr) genes contain intervening sequences of variable sequence and length ranging in size from 11 to 21 bp. However, two features are conserved in all plant pre-tRNA(Tyr) introns: they possess a uridine and less frequently an adenosine at the 5' boundary and can adopt similar intron secondary structures in which an extended anticodon helix of 4-5 bp is formed by base-pairing between nucleotides of the intron and the anticodon loop. In order to elucidate the potential role of the highly conserved uridine at the first intron position, we have replaced it by all other nucleosides in an Arabidopsis pre-tRNA(Tyr) and have studied in wheat germ extract its effect on splicing and on conversion of U to psi in the GpsiA anticodon. Furthermore, we discuss the putative acquisition of tRNA(Tyr) introns at an early step of evolution after the separation of Archaea and Eucarya.

Anticodon↗

PubMed1997

Activation of a cryptic splice-site in intron 24 leads to the formation of apolipoprotein B-27.6.

Apo B expression is confined to the intestine and liver, and its secretion from these tissues is dependent on the expression of a lipid transfer protein, microsomal triglyceride transfer protein (MTP). Previously, we reported a model system for the study of apolipoprotein (apo B) biogenesis using heterologous expression in COS cells (Patel SB, Grundy SM. J. Lipid Res. 1995;36:2090-2103). We now report the characterization of the effects of a T-->C transition in the splice-site at +2 of intron 24 previously reported by Talmud et al. (J. Lipid Res. 1994;35:468-77). Using our heterologous expression system, we show that the mutation led to aberrant processing of intron 24, but normal processing of intron 25. The resultant translation of this mutant mRNA produced a truncated apo B protein of the size of apo B-27.6. Reverse transcription, polymerase chain reaction and sequencing of the amplified products were used to show that a cryptic donor splice-site within intron 24 was utilized, resulting in the generation of a novel hydrophilic 29 amino acid carboxyl-terminal tail. Co-expression of apo B-27.6 with microsomal triglyceride transfer protein (MTP) showed that this protein could bind MTP and resulted in the secretion of a lipoprotein particle with a buoyant density in the range 1.16-1.25 g/ml. These results indicate that this splice-site mutation leads to an activation of a downstream cryptic splice-site within intron 24, causing an insertion of 40 bases of intron 24 sequences into the mature RNA. This leads to a frame-shift of translation resulting in addition of 29 new amino acids at the carboxyl-terminus, before an in-frame stop translation codon is encountered, truncating the apo B at B-27.6.

Alternative Splicing↗

PubMed2002

Characterization of the C-terminal DNA-binding/DNA endonuclease region of a group II intron-encoded protein.

Group II intron retrohoming occurs by a mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded reverse transcriptase uses a C-terminal DNA endonuclease activity to cleave the opposite strand and then uses the cleaved 3' end as a primer for reverse transcription of the inserted intron RNA. Here, we characterized the C-terminal DNA-binding/DNA endonuclease region of the LtrA protein encoded by the Lactococcus lactis Ll.LtrB intron. This C-terminal region consists of an upstream segment that contributes to DNA binding, followed by a DNA endonuclease domain that contains conserved sequence motifs characteristic of H-N-H DNA endonucleases, interspersed with two pairs of conserved cysteine residues. Atomic emission spectroscopy of wild-type and mutant LtrA proteins showed that the DNA endonuclease domain contains a single tightly bound Mg(2+) ion at the H-N-H active site. Although the conserved cysteine residue pairs could potentially bind Zn(2+), the purified LtrA protein is active despite the presence of only sub-stoichiometric amounts of Zn(2+), and the addition of exogenous Zn(2+) inhibits the DNA endonuclease activity. Multiple sequence alignments identified features of the DNA-binding region and DNA endonuclease domain that are conserved in LtrA and related group II intron proteins, and their functional importance was demonstrated by unigenic evolution analysis and biochemical assays of mutant LtrA protein with alterations in key amino acid residues. Notably, deletion of the DNA endonuclease domain or mutations in its conserved sequence motifs strongly inhibit reverse transcriptase activity, as well as bottom-strand cleavage, while retaining other activities of the LtrA protein. A UV-cross-linking assay showed that these DNA endonuclease domain mutations do not block DNA primer binding and thus likely inhibit reverse transcriptase activity either by affecting the positioning of the primer or the conformation of the reverse transcriptase domain.

Amino Acid Motifs↗

PubMed1991

Proto-splice site model of intron origin.

It is proposed that nuclear pre-mRNA introns (classical introns) were first generated as by-products during the evolution of alternative splicing. They were formed whenever two splice sites within the coding sequence of ancestral genes were used at a frequency that removed the coding constraint from the intervening sequence. Once introns had evolved, it is suggested that they were spread by the splicing machinery which inserted them into proto or cryptic-splice sites of other genes by reverse splicing, so giving rise to genes that have introns yet are not alternatively spliced. It is argued that 5' and 3' splice sites evolved from common ancestral splice sites, referred to as proto-splice sites, that were bidirectional and had a core consensus sequence of C or A, A, G, R, which remains today as the immediate flanking sequence of most introns. The ancestral splicing machinery, although inefficient, would have been capable of generating vast mRNA diversity by splicing between proto-splice sites. Natural selection would be expected to have preserved mutations that increased the amounts of advantageously spliced mRNA. It is argued that this process drove the evolution of present 5' and 3' splice sites from a subset of proto-splice sites and also drove the evolution of a more efficient splicing machinery. The positions of most introns that evolved directly from the coding sequence would be expected to correlate with protein structure.

Animals↗

PubMed1992

Mouse U14 snRNA is a processed intron of the cognate hsc70 heat shock pre-messenger RNA.

U14 snRNA is a small nucleolar RNA species essential for eukaryotic pre-rRNA processing. We have previously shown that the mouse U14 snRNA genes are positioned within introns 5, 6, and 8 on the coding strand of the constitutively expressed cognate hsc70 heat shock gene. This genomic organization suggested the possibility that U14 snRNAs are transcribed as part of the hsc70 pre-mRNA and then excised from the intron to yield mature U14 snRNA species. To test this hypothesis directly, we have microinjected Xenopus oocytes with hsc70 pre-mRNA transcripts possessing intron 5 and the encoded U14 snRNA sequence. Processing results demonstrate that, in addition to the splicing of upstream and downstream exons, a mature 87 nt U14 snRNA is excised from the intron. Accurate excision of U14 snRNA from hsc70 intron 5 can occur in the absence of splicing. These results demonstrate a biosynthetic pathway for an snRNA species and provide a novel example of a eukaryotic pre-mRNA intron that is processed to produce a stable, biologically functional RNA species.

Amino Acid Sequence↗

PubMed2000

Analysis of tyrosine hydroxylase gene transcription using an intron specific probe.

The nuclear run-on assay is the most commonly used technique to determine transcription rates of specific genes such as tyrosine hydroxylase. Its application to studies in the nervous system is problematic, however, as a result of limitations in sensitivity and the loss of anatomical integrity. We observed that the relative levels of tyrosine hydroxylase intron 2-containing RNA using a ribonuclease protection assay in the adrenal medulla changed in response to pharmacological treatments consistently with changes shown by the nuclear run-on assay. Our results indicate that measures of tyrosine hydroxylase primary transcript levels offer an alternative to the nuclear run-on assay and validate the application of intron-specific in situ hybridization as a means of assessing the relative transcriptional activity of the tyrosine hydroxylase gene. Similar quantitative results were obtained using intron-specific in situ hybridization with oligonucleotide probes specific for rat tyrosine hydroxylase intron 2. Furthermore, we observed that intron-specific in situ hybridization could be used to measure tyrosine hydroxylase transcription rates in the locus coeruleus, providing resolution at the level of single neurons. Thus, measuring the levels of tyrosine hydroxylase intron 2 provides a sensitive measure of tyrosine hydroxylase transcription rate that can be applied to the study of brain catecholaminergic neurons.

Adrenal Medulla↗