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At least 1,369 records · Page 76Linked to original sources
PubMed1998

A map of the binding site for catalytic domain 5 in the core of a group II intron ribozyme.

Group II introns are ribozymes with a complex tertiary architecture that is of great interest as a model for RNA folding. Domain 5 (D5) is a highly conserved region of the intron that is considered one of the most critical structures in the catalytic core. Despite its central importance, the means by which D5 interacts with other core elements is unclear. To obtain a map of potential interaction sites, dimethyl sulfate was used to footprint regions of the intron that are involved in D5 binding. These studies were complemented by measurements of D5 binding to a series of truncated intron derivatives. In this way, the minimal region of the intron required for strong D5 association was defined and the sites most likely to represent thermodynamically significant positions of tertiary contact were identified. These studies show that ground-state D5 binding is mediated by tertiary contacts to specific regions of D1, including a tetraloop receptor and an adjacent three-way junction. In contrast, D2 and D3 are not found to stabilize D5 association. These data highlight the significance of D1-D5 interactions and will facilitate the identification of specific tertiary contacts between them.

Base Sequence↗
PubMed2001

Mechanism of maturase-promoted group II intron splicing.

Mobile group II introns encode reverse transcriptases that also function as intron-specific splicing factors (maturases). We showed previously that the reverse transcriptase/maturase encoded by the Lactococcus lactis Ll.LtrB intron has a high affinity binding site at the beginning of its own coding region in an idiosyncratic structure, DIVa. Here, we identify potential secondary binding sites in conserved regions of the catalytic core and show via chemical modification experiments that binding of the maturase induces the formation of key tertiary interactions required for RNA splicing. The interaction with conserved as well as idiosyncratic regions explains how maturases in some organisms could evolve into general group II intron splicing factors, potentially mirroring a key step in the evolution of spliceosomal introns.

Aldehydes↗
PubMed2002

An inserted region of leucyl-tRNA synthetase plays a critical role in group I intron splicing.

Yeast mitochondrial leucyl-tRNA synthetase (LeuRS) binds to the bI4 intron and collaborates with the bI4 maturase to aid excision of the group I intron. Deletion analysis isolated the inserted LeuRS CP1 domain as a critical factor in the protein's splicing activity. Protein fragments comprised of just the LeuRS CP1 region rescued complementation of a yeast strain that expressed a splicing-defective LeuRS. Three-hybrid analysis determined that these CP1-containing LeuRS fragments, ranging from 214 to 375 amino acids, bound to the bI4 intron. In each case, interactions with only the LeuRS protein fragment specifically stimulated bI4 intron splicing activity. Substitution of a homologous CP1 domain from isoleucyl-tRNA synthetase or mutation within the LeuRS CP1 region of the smallest protein fragment abolished RNA binding and splicing activity. The CP1 domain is best known for its amino acid editing activity. However, these results suggest that elements within the LeuRS CP1 domain also play a novel role, independent of the full-length tRNA synthetase, in binding the bI4 group I intron and facilitating its self-splicing activity.

Blotting, Western↗
PubMed1995

Constraints on intron evolution in the gene encoding the myosin alkali light chain in Drosophila.

Interspecific comparisons of intron sequences reveal conserved blocks of invariant nucleotides and several other departures from the strictly neutral model of molecular evolution. To distinguish the past action of evolutionary forces in introns known to have regulatory information, we examined nucleotide sequence variation at 991 sites in a random sample of 16 Drosophila melanogaster alleles of the gene encoding the myosin alkali light chain (Mlc1). The Mlc1 gene of D. melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. Analyses of these data reveal that introns 4 and 5, which flank the alternatively spliced exon 5, have reduced levels of both intraspecific polymorphism and interspecific divergence relative to intron 3. No polymorphism was observed in any of the exons examined in D. melanogaster. A genealogical analysis clearly demonstrates the occurrence of intragenic recombination in the ancestral history of Mlc1. Recombination events are estimated to be 13 times more likely than mutation events over the span of the sequenced region. Although there is little evidence for pairwise linkage disequilibrium in the Mlc1 region, higher order disequilibrium does seem to be present in the 5' half of the portion of the gene that was examined. Predictions of the folding free energy of the pre-mRNA reveal that sampled alleles have a significantly higher (less stable) free energy than do randomly permuted sequences. These results are consistent with the hypothesis that introns surrounding an alternatively spliced exon are subjected to additional constraints, perhaps due to specific aspects of secondary structure required for appropriate splicing of the pre-mRNA molecule.

Alternative Splicing↗
PubMed2002

How intron splicing affects the deletion and insertion profile in Drosophila melanogaster.

Studies of "dead-on-arrival" transposable elements in Drosophila melanogaster found that deletions outnumber insertions approximately 8:1 with a median size for deletions of approximately 10 bp. These results are consistent with the deletion and insertion profiles found in most other Drosophila pseudogenes. In contrast, a recent study of D. melanogaster introns found a deletion/insertion ratio of 1.35:1, with 84% of deletions being shorter than 10 bp. This discrepancy could be explained if deletions, especially long deletions, are more frequently strongly deleterious than insertions and are eliminated disproportionately from intron sequences. To test this possibility, we use analysis and simulations to examine how deletions and insertions of different lengths affect different components of splicing and determine the distribution of deletions and insertions that preserve the original exons. We find that, consistent with our predictions, longer deletions affect splicing at a much higher rate compared to insertions and short deletions. We also explore other potential constraints in introns and show that most of these also disproportionately affect large deletions. Altogether we demonstrate that constraints in introns may explain much of the difference in the pattern of deletions and insertions observed in Drosophila introns and pseudogenes.

Animals↗
PubMed1995

Splicing mutations of the polycystic kidney disease 1 (PKD1) gene induced by intronic deletion.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease which frequently results in renal failure. The major ADPKD gene, polycystic kidney disease 1 (PKD1), has recently been identified. In an attempt to understand better the aetiology of this disorder we have searched for mutations in the PKD1 gene. Analysis of three regions in the 3' part of the gene has revealed two mutations that occur by a novel mechanism. Both mutations are deletions (of 18 or 20 bp) within the same 75 bp intron and although these deletions do not disrupt the splice donor or acceptor sites at the boundary of the intron, they nevertheless result in aberrant splicing. Two different transcripts are produced in each case; one includes the deleted intron while the other has a 66 bp deletion due to activation of a cryptic 5' splice site. No normal product is generated from the deleted gene. Aberrant splicing probably occurs because the deleted intron is too small for spliceosome assembly using the authentic splice sites; this mechanism has previously only been described from in vitro studies of vertebrate genes. A 9 bp direct repeat has been identified within the intron, which probably facilitated deletion by promoting misalignment of sequence. The possible phenotypic implications of producing more than one aberrant PKD1 transcript in these cases are discussed.

Adult↗
PubMed2003

Messenger RNA surveillance and the evolutionary proliferation of introns.

The mechanisms responsible for the proliferation and subsequent stabilization of introns within the eukaryotic lineage have remained elusive. In the early stages of eukaryotic evolution, most introns may have been mildly deleterious at the time of insertion, but enough of them eventually acquired integral roles in transcript processing that few eukaryotic species can any longer survive without them. We suggest that the proliferation of spliceosomal introns was facilitated by the evolution of nonsense-mediated decay, an ancient and (in many cases) intron-dependent mechanism for eliminating aberrant mRNA molecules resulting from errors in transcription and splicing and from mutations at the DNA level. The spatial distribution of introns, as revealed by whole-genome analysis, is consistent with expectations for a model in which maximum protective coverage of a gene stochastically evolves over time.

Alternative Splicing↗
PubMed2006

Estimating the neutral rate of nucleotide substitution using introns.

Evolutionary biologists frequently rely on estimates of the neutral rate of evolution when characterizing the selective pressure on protein-coding genes. We introduce a new method to estimate this value based on intron nucleotide substitutions. The new method uses a metascript model that considers alternative splicing forms and an algorithm to pair orthologous introns, which we call Introndeuce. We compare the intron method with a widely used method that uses observed substitutions in synonymous coding nucleotides, by using both methods to estimate the neutral rate for human-dog and mouse-rat comparisons. The estimates of the 2 methods correlate strongly (r(S) = 0.75), but cannot be considered directly equivalent. We also investigate the effect of alignment error and G + C content on the variance in the intron method: in both cases there is an effect, and it is species-pair specific. Although the intron method may be more useful for shorter evolutionary distances, it is less useful at longer distances due to the poor alignment of less-conserved positions.

Algorithms↗
PubMed1997

Enhanced restriction site mutation (RSM) analysis of 1,2-dimethylhydrazine induced mutations, using endogenous p53 intron sequences.

The restriction site mutation (RSM) assay was used to study the mutational sensitivities of three target regions of the murine p53 gene. The non-coding intron 6 target region was compared with the coding regions exon 4 and exon 5 with respect to their relative sensitivity to the induction of mutations by 1,2-dimethylhydrazine (DMH). Our results demonstrated that the majority of induced mutations detected were in the intron 6 gene region. A total of 15 enzyme-resistant restriction sites were detected in DMH treated mice, nine of these in the intron 6 region, four in the exon 4 region and two in the exon 5 region. The elevated sensitivity of the intron 6 region was exemplified by our detection of spontaneous mutations in this region; two resistant restriction sites were detected in untreated animals. No spontaneous mutations were detected in either of the exon sequences studied here, nor have any been detected in exon targets in our previous in vivo RSM analyses. The mutations induced by DMH were mostly GC-->AT transitions, as were the spontaneous mutations identified. The mutation frequencies calculated by the inclusion of a mutant internal standard (MIS) in the RSM method, revealed that the non-coding intron 6 region and the exon 4 region had a 10-fold higher mutation frequency than the exon 5 region. This heterogenous distribution of mutations and their differential mutation frequencies, were probably a consequence of the greater selection in the coding regions in p53 function. However, the actual mechanism of differential mutation induction is, as yet, to be defined.

1,2-Dimethylhydrazine↗
PubMed1987

Euglena gracilis chloroplast DNA: the untranslated leader of tufA-ORF206 gene contains an intron.

Structural features of a dicistronic 1.95 kb mRNA coding for the chloroplast specific elongation factor Tu and ORF206 are described. The unspliced pre-mRNA is composed of 2562 nucleotides and undergoes four splicing events which remove a total of 606 nucleotides. The first intron splits the untranslated leader, two introns dissect the tufA coding region and the forth intron is within ORF206, which codes for a putative protein that is to 34% homologous with the putative protein of chloroplast ORF184 of tobacco. Introns neither belong to group I nor II, and 5' and 3' intron boundaries do not follow consensus sequences. Potential ribosome binding sites are located 58 and 32 positions upstream of the tufA and ORF206 start codon, respectively.

Amino Acid Sequence↗
PubMed1987

Expression of intron-containing C. elegans heat shock genes in mouse cells demonstrates divergence of 3' splice site recognition sequences between nematodes and vertebrates, and an inhibitory effect of heat shock on the mammalian splicing apparatus.

Splicing of a pair of intron-containing heat shock genes from Caenorhabditis elegans has been studied in transfected mouse cells. The hsp16-1 and hsp16-48 genes of C. elegans encode 16,000 Da heat shock polypeptides. Each gene contains a short intron of 52 (hsp16-1) or 55 (hsp16-48) base pairs. When these genes were introduced into mouse cells, they were efficiently induced following heat shock, but splicing of the introns was abnormal. In mouse cells, cleavage of the hsp16 transcripts occurred at the correct 5' splice sites, but the 3' splice sites were located at AG dinucleotides downstream of the correct sites. This aberrant splicing was not solely due to the small size of the C. elegans introns, since a hsp16-1 gene containing an intron enlarged by tandem duplication showed exactly the same splicing pattern. The mouse cells thus seem to be unable to recognize the natural 3' splice sites of the C. elegans transcripts. The efficiency of splicing was greatly reduced under heat shock conditions, and unspliced transcripts accumulated in the nucleus. During a subsequent recovery period at 37 degrees C, these transcripts were spliced and transported to the cytoplasm.

Animals↗
PubMed1988

The intron requirement for immunoglobulin gene expression is dependent upon the promoter.

Transfection assays were used to assess the need for an intron in order to obtain expression of cytoplasmic immunoglobulin mu mRNA. An intron is required when transcription is driven by an immunoglobulin promoter/enhancer combination, although this requirement is not specific for a particular intron. However, this need for an intron is dependent upon the promoter used. Whilst an intron is required in the case of immunoglobulin or beta-globin promoters, it is not in the case of cytomegalovirus or heat-shock promoters. The data point to a connection between the promoter and RNA processing or export.

Enhancer Elements, Genetic↗
PubMed1988

All human tRNATyr genes contain introns as a prerequisite for pseudouridine biosynthesis in the anticodon.

Two synthetic oligonucleotides, one specific for the 5' exon, the other spanning the splice junction, were used to show that (a) the human haploid genome contains at least 12 independent gene loci for tRNATyr, and (b) that all of them carry an intron. From one of the cloned human tRNATyr genes (pHtT1) the 20 bp intron was deleted to generate pHtT1 delta. Homologous in vitro transcription, fingerprint analyses of the products and elucidation of their nucleoside composition revealed that the pseudouridine (psi 35) in the center of the anticodon of tRNATyr was synthesized in the intron-containing precursor whereas this U to psi modification did not take place in precursors or mature tRNATyr derived from pHtT1 delta. On the basis of these results and of studies from other laboratories we suggest that the evolutionary pressure for maintaining introns in eukaryotic tRNAsTyr is this strict intron-requirement for psi 35 synthesis. Taking into account that all eukaryotic cytoplasmic tRNAsTyr contain a psi 35 we discuss here a special need for this modified nucleoside in stabilizing codon-anticodon interactions involving (a) classical base pairing upon translation of tyrosine codons and (b) unconventional interactions during UAG amber codon suppression by tRNATyrG psi A in eukaryotic cells.

Anticodon↗
PubMed1989

Evidence for a class of very small introns in the gene for hypoxanthine-guanine phosphoribosyltransferase in Schistosoma mansoni.

The single copy gene for the hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) of the parasitic trematode, Schistosoma mansoni, contains seven introns, the first four of which are only 31, 33, 42, and 32 bases in length. These are the smallest introns ever discovered in a non-viral nuclear gene coding for protein. These very small introns possess the canonical GT...AG splice site sequences but lack the branching sequence, the secondary structure, and the minimum size of approximately 50 bases believed to be required for the splicing of eucaryotic mRNA precursors. Evidently, a somewhat different splicing mechanism for the transcripts of these very small introns is necessary. Their discovery within the genes of helminths raises theoretical considerations for the evolution of introns in eucaryotes.

Amino Acid Sequence↗
PubMed1990

Identification of tissue specific nuclear proteins: DNA sequence and protein binding regions in the T cell receptor beta J-C intron.

We have determined the DNA sequences in the J2-C2 intron of the T cell receptor (TCR) beta gene and analyzed nuclear proteins binding to this region. Previously, we identified two tissue-specific DNase I hypersensitive regions, potential regulatory regions, in the J-C intron. The DNA sequence of the J2-C2 intron revealed that both DNase I hypersensitive regions have similar DNA sequences, suggesting that these regions are evolutionarily conserved. We have also identified tissue-specific nuclear-protein binding regions downstream of the DNase hypersensitive regions. Although transcriptional enhancer activity was not observed in the hypersensitive regions or the adjacent protein binding regions in the J-C intron, our findings suggest that the TCR-beta J-C intron may contain some other type of regulatory element.

Animals↗
PubMed1990

Group II intron RNA-catalyzed recombination of RNA in vitro.

We report the first evidence for a novel reaction mediated by the self-splicing yeast mitochondrial group II intron bl1; the site-specific recombination of RNA molecules in vitro. Upon incubation of the intron lariat with two different RNAs, each harbouring a short sequence complementary to exon binding site 1 (EBS1) of the intron, novel recombined RNAs are formed. As a result of this intron-mediated shuffling of gene segments, the 5' part of RNA1 is ligated to the 3' part of RNA2 and, reciprocally, the 5' part of RNA2 to the 3' part of RNA1. Sequence analysis of the recombinant junction shows that the site of recombination is precisely located 3' to intron binding site 1 (IBS1). The hypothesized mechanism of recombination involves exchange of RNA 5' parts after the first step of a reverse splicing reaction. The possible role of this mechanism in vivo and during prebiotic evolution is discussed.

Base Sequence↗
PubMed1990

Information content of Caenorhabditis elegans splice site sequences varies with intron length.

A database of sequences of 139 introns from the nematode Caenorhabditis elegans was analyzed using the information measure of Schneider et al. (1986) J. Mol. Biol. 128: 415-431. Statistically significant information is encoded by at least the first 30 nt and last 20 nt of C. elegans introns. Both the quantity and the distribution of information in the 5' splice site sequences differs between the typical short (length less than 75 nt) and rarer long (length greater than 75 nt) introns, with the 5 sites of long introns containing approximately one bit more information. 3' splice site sequences of long and short C. elegans introns differ significantly in the region between -20 and -10 nt.

Animals↗
PubMed1991

Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells.

A 2.4 kb fragment of hCMV (Towne strain), containing the 5' end of the major immediate-early gene, has been cloned, sequenced, and used to construct a series of mammalian cell expression plasmids. The effects of regulatory regions present on this fragment were assessed using human glycoproteins as reporter molecules. We compared secreted levels of Factor VIII, t-PA, and HIV-1 envelope glycoproteins in cells transfected with plasmids in which intron A of the immediate-early gene was present or absent. Secretion of several glycoproteins was significantly higher when cells were transfected with intron A-containing plasmids. Mutation of three basepairs in the strong nuclear factor 1 (NF1) binding site in intron A led to reduced transient expression levels, but not to the level observed in the absence of intron A. Reduced expression from NF1 mutant plasmids was roughly correlated with reduced binding in vitro of NF1 proteins to a synthetic oligonucleotide containing the mutation. The evidence indicates that sequences in intron A positively regulate expression from the hCMV immediate-early enhancer/promoter in transformed monkey kidney cells.

Animals↗