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At least 145 records · Page 8Linked to original sources

PubMed2003

Is the human dystrophin gene's intron structure related to its intron instability?

OBJECTIVE: To study the human dystrophin gene molecular deletion mechanism, we analyzed breakpoint regions within junction fragments of deletion-type patients and investigated whether the dystrophin gene's intron structure might be related to intron instability. METHODS: Junction fragments corresponding to exon 46 and 51 deletions were cloned. The breakpoint regions were sequenced, and the features of introns with available Genebank sequences were analyzed. RESULTS: An analysis of junction fragment sequences corresponding to exon 46 and 51 deletions showed that all 5' and 3' breakpoints are located within repeat sequences. No small insertions, small deletions, or point mutations are located near the breakpoint junctions. By analyzing the secondary structure of the junction fragments, we demonstrated that all junction fragment breakpoints are located in non-matching regions of single-stranded hairpin loops. A high concentration of repetitive elements is found to be a key feature of many dystrophin introns. In total, 34.8% of the overall dystrophin intron sequences is composed of repeat sequences. CONCLUSION: Repeat elements in many dystrophin gene introns are the key to their structural bases and reflect intron instability. As a result of the primary DNA sequences, single-stranded hairpin loops form, increasing the instability of the gene, and forming the base for breaks in the DNA. The formation of the single-stranded hairpins can result in reattachment of two different breakpoints, producing a deletion.

Dystrophin↗

PubMed1982

Critical sequences within mitochondrial introns: pleiotropic mRNA maturase and cis-dominant signals of the box intron controlling reductase and oxidase.

We have established the DNA sequence of nine yeast mutants that prevent the expression either of the split cytochrome b gene alone (five mutants) or of two split genes, the cytochrome b gene and the cytochrome oxidase subunit I gene (four mutants). All the mutations analyzed are localized in intron 14 of the cob-box gene. We have extended the concept of the intron-encoded mRNA maturase, already described for intron 12, to the intron 14, and have adduced evidence that this box7 pleiotropic maturase is involved in the splicing of two distant gene transcripts. Such a process may constitute a regulatory mechanism that coordinates the expression of two structurally nonhomologous genes encoding two metabolically related enzymes. Analyses of cis-dominant mutations reveal the role of signal sequences in the recognition of the intron RNA sequences to be excised. These signal sequences are localized near the exon-intron boundaries (box1), or quite distant from the splicing sites, either in the blocked reading frame (box2) or in the open reading frame (box9) of the intron. We believe that for the last sequence, a ribosomal recognition of the box9 signal could be involved in a regulatory mechanism of the splicing of the pre-mRNA.

Base Sequence↗

PubMed1984

A minimal intron length but no specific internal sequence is required for splicing the large rabbit beta-globin intron.

We constructed rabbit beta-globin genes with deletions in the large intron, extending from the midpoint toward the 5' or 3' splice sites. Analysis of transcripts in transformed HeLa cells showed that six 5' proximal intron nucleotides allowed normal splicing. Correct splicing at the 3' splice site required 12 or more 3' proximal intron nucleotides; optimal efficiency required 24 nucleotides. Remarkably, a mini-intron comprising six 5' and 24 3' intron nucleotides gave no correctly spliced transcripts; extending the miniintron with polyoma or pBR322 fragments to 80 or more nucleotides restored normal splicing. Thus other than in yeast nuclear genes, no specific internal intron sequences appear to be needed but a minimal intron length is important.

Animals↗

PubMed1986

Exon/intron organization of the chicken type II procollagen gene: intron size distribution suggests a minimal intron size.

Overlapping genomic clones have been isolated that contain the alpha chain and COOH-terminal propeptide coding regions of the chicken type II procollagen gene. All type II procollagen exon sequences present in these clones have been identified and mapped by DNA sequencing. These include 43 exons coding for the alpha-chain triple helix, 1 exon coding for the junction between the COOH-terminal propeptide and the alpha-chain region, and 3 exons coding for the COOH-terminal propeptide and 3' noncoding sequences. With the exception of one additional intron between 2 exons coding for amino acids 568-585 and 586-603, exon-intron boundaries have been conserved when compared with genes for all other characterized genes for fibrillar collagens. The chicken type II procollagen gene differs from most other collagen genes in having introns of considerably smaller average size. The size distribution of the introns suggests that approximately equal to 80 base pairs may be a minimal functional size for introns in this gene. This size of intron may be necessary in a gene with a very large number of small exons to prevent aberrant splicing from removing exon sequence together with intron sequence.

Animals↗

PubMed1993

Self-splicing of a Podospora anserina group IIA intron in vitro. Effects of 3'-terminal intron alterations on cleavage at the 5' and 3' splice site.

A shortened derivative of the group IIA intron from the mitochondrial cytochrome-c-oxidase subunit I gene (COI I1) of the ascomycete Podospora anserina can undergo self-splicing in vitro. When compared to self-splicing group IIB introns from yeast mitochondria (aI5c, bI1) the autocatalytic reaction shows a lower efficiency and 5' cleavage takes place predominantly by hydrolysis. In order to test the influence on reaction efficiency and mode of 5' cleavage of the long peripheral structure of domain VI (dVI) we generated mutant Podospora introns that have different structural forms of shortened dVI. Our results show that: (1) in general the size and structure of dVI distal from the branch site is essential for 5' transesterification and influences the efficiency of the second splicing step; (2) 5' transesterification as well as the complete self-splicing reaction is more efficient when the structure of dVI is adapted to that of yeast group IIB introns. Moreover, our data indicate that the postulated gamma-gamma' tertiary interaction is also functional for group IIA introns. A weakening or disruption of this interaction in the Podospora intron leads to a greatly reduced cleavage at the 3' splice site and to a selection of cryptic sites downstream in the 3' exon that almost exclusively restore the strong wild-type gamma-gamma' pairing. The so-called "guide" interaction seems to support the selection of 3' cleavage sites but is of secondary importance in relation to the gamma-gamma' interaction.

Ascomycota↗

PubMed1993

Monitoring of the cooperative unfolding of the sunY group I intron of bacteriophage T4. The active form of the sunY ribozyme is stabilized by multiple interactions with 3' terminal intron components.

We have studied the mechanism by which the 3' terminal domain of the sunY intron of bacteriophage T4 activates the group I ribozyme core of this intron, from which it is separated by some 800 nucleotides. As shown by monitoring either UV absorbance or self-splicing reaction kinetics as a function of temperature, intron transcripts undergo highly cooperative unfolding/inactivation upon heating: the two methods yield similar estimates of the thermodynamic parameters associated with this process. Such cooperativity makes it possible in turn to assess the energetic contribution of specific interactions to the overall structure, by comparing the sensitivity to heat inactivation of molecules carrying various nucleotide substitutions. By combining this approach with chemical modification, we have probed several proven or putative interactions between the core and 3' terminal domain of the intron and conclude that the role of the 3' terminal domain is to stabilize the active form of the ribozyme. Interestingly, the P9.0 interaction, which brings 3' terminal nucleotides next to the core site that binds the guanosine cofactor of the self-splicing reaction, is now shown to be composed in fact of two distinct pairings. An isolated base-pair (P9.0a), involving a residue located only six nucleotides upstream of the 3' splice site, participates in the stabilization of the ribozyme and appears to persist during the second stage of self-splicing (exon ligation). In contrast, formation of the previously demonstrated P9.0b pairing, which involves the two penultimate intron nucleotides, contributes no additional stability and results in no detectable rearrangement of the core structure. Implications for the concept of a static ribozyme are discussed in the light of a slightly revised three-dimensional model of the sunY intron.

Bacteriophage T4↗

PubMed1990

Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation.

Agrobacterium tumefaciens is a commonly used tool for transforming dicotyledonous plants. The underlying mechanism of transformation however is not very well understood. One problem complicating the analysis of this mechanism is the fact that most indicator genes are already active in Agrobacterium, thereby preventing the precise determination of timing and localisation of T-DNA transfer to plant cells. In order to overcome this obstacle a modified prokaryotic indicator gene was constructed. The expression of this indicator gene and its use in analysing early events in Agrobacterium-mediated plant transformation are described. A portable intron, derived from a plant intron, was introduced into the beta-glucuronidase (GUS) gene. In transgenic plants containing this chimaeric gene the intron is spliced efficiently, giving rise to GUS enzymatic activity. Mapping of the splice junction indicates the exact removal of the intron. No GUS activity is detected in agrobacteria containing this construct due to the lack of a eukaryotic splicing apparatus in prokaryotes. Early phases after transformation of Arabidopsis cotyledon explants were analysed using this GUS-intron chimaeric gene showing that as early as 36 h after Agrobacterium infection significant GUS activity is detected. In vivo GUS staining of transformed cells clearly shows that quickly proliferating calli expressing GUS activity are formed, mainly at the cut surface. Minor transformation events occur however throughout the whole cotyledon. These data indicate that Agrobacterium-mediated T-DNA transfer to plants is much more efficient than has been judged from experiments where selection is applied immediately. The intron-containing GUS gene can be used as an optimised marker gene in transient and stable transformation experiments.

Base Sequence↗

PubMed1991

Intron enhancement of gene expression and the splicing efficiency of introns in maize cells.

The inclusion of the alcohol dehydrogenase 1-S(Adh 1-S) intron 1 in the transcription unit of maize gene constructs has been shown to increase gene expression in cultured maize cells. We have extended these studies with Adh1-S intron 1 using the firefly luciferase, Escherichia coli beta-glucuronidase and chloramphenicol acetyltransferase reporter genes adjoined to different plant promoters and find enhancement of transient gene expression in all cases but one. We also show that the enhancement phenomenon can be mediated by the third intron of the maize actin gene. In all cases tested, the inclusion of an intron results in increased levels of steady-state RNA. The degree of enhancement depends on the exon sequences flanking the intron; flanking exons also influence the efficiency of intron splicing. Unexpectedly, unspliced RNAs accumulate during the transient assay.

Alcohol Dehydrogenase↗

PubMed1999

A Group I intron in the nuclear small subunit rRNA gene of Cryptendoxyla hypophloia, an ascomycetous fungus: evidence for a new major class of Group I introns.

The ascomycetous fungus Cryptendoxyla hypophloia contains an insertion of 433 base pairs in the genes encoding nuclear small subunit ribosomal RNA. Secondary structure analyses of the insert reveal characteristics indicative of a Group I intron, including elements P, Q, R, and S; however, the sequences of these conserved regions deviate significantly from recognized consensus sequences for Group I introns. Principal-components analysis, based on 79 nucleotide positions from the conserved core sequences of 93 Group I introns, identified 17 introns similar to that of C. hypophloia. This grouping, which includes inserts from phylogenetically diverse organisms, cannot readily be classified in any previously recognized major group of Group I introns. We propose the creation of a new group, IE, to accommodate these sequences, and discuss the evolutionary relationships between group IE and other major groups of Group I introns.

Ascomycota↗

PubMed1998

Splicing of the mitochondrial group-II intron rl1: conserved intron-exon interactions diminish splicing efficiency.

The mitochondrial intron rI1 is a self-splicing group-II intron of algal mitochondria that can be transferred into chloroplasts from the green alga Chlamydomonas reinhardtii for in vivo investigations (Herdenberger et al. 1994). Thus, rI1 is a suitable system to compare in vitro and in vivo RNA processing. Interestingly, rI1 shows correct RNA splicing, although typical cis-acting exon-sequences (IBS2, delta) of group-II introns are lacking. In order to examine the effect of these exon-intron interactions on splicing, we introduced the endogenous mitochondrial IBS2 sequence in order to produce optimal IBS2-EBS2 base pairing. In addition, the first nucleotide of the 3'exon (delta') was substituted to create an optimal delta-delta' interaction. Neither of the two mutations, nor a combination of both, had any effect on the precision of the splice-site selection. Unexpectedly, introduction of IBS2 led to a reduction in the efficiency of the second splicing step in vitro but not in vivo. These findings lead us to conclude that trans-acting factors are present in vivo to optimize splicing efficiency. The possibility is discussed that these factors may, for example, stabilize tertiary intron structures that are a prerequisite for correct RNA processing. Furthermore, our data indicate that similar trans-acting factors promote correct intron splicing in chloroplasts and mitochondria.

Alternative Splicing↗

PubMed1989

Reverse self-splicing of the tetrahymena group I intron: implication for the directionality of splicing and for intron transposition.

Using short oligoribonucleotides as ligated exon substrates, we show that splicing of the Tetrahymena rRNA group I intron is fully reversible in vitro. Incubation of ligated exon RNA with linear intron produces a molecule in which the splice site sequences of the precursor are reformed. Reversal of self-splicing is favored by high RNA concentration, high magnesium and temperature, and the absence of guanosine. 5' exon sequences that can pair with the internal guide sequence of the intron are required, whereas 3' exon sequences are not essential. Integration of the intron into ligated exon substrates that have the ability to form stem-loop structures is reduced at least one order of magnitude over short, unstructured substrates. We propose that the formation of these structures helps drive splicing in the forward direction. We also show that the Tetrahymena intron can integrate into a beta-globin transcript. This has implications for transposition of group I introns.

Animals↗

PubMed1992

The neurospora CYT-18 protein suppresses defects in the phage T4 td intron by stabilizing the catalytically active structure of the intron core.

The Neurospora CYT-18 protein, a tyrosyl-tRNA synthetase, which functions in splicing group I introns in mitochondria, promotes splicing of mutants of the distantly related bacteriophage T4 td intron. In an in vivo assay, wild-type CYT-18 protein expressed in E. coli suppressed mutations in the td intron's catalytic core. CYT-18-suppressible mutations were also suppressed by high Mg2+ or spermidine in vitro, suggesting they affect intron structure. Both the N- and C-terminal domains of CYT-18 are required for efficient splicing, but CYT-18 with a large C-terminal truncation retains some activity. Our results indicate that CYT-18 interacts with conserved structural features of group I introns, and they provide direct evidence that a protein promotes splicing by stabilizing the catalytically active structure of the intron RNA.

Base Sequence↗

PubMed2006

An alternative intron-exon pairing scheme implied by unexpected in vitro activities of group II intron RmInt1 from Sinorhizobium meliloti.

RmInt1 is a mobile group II intron which interrupts ISRm2011-2, another mobile element from the bacterium Sinorhizobium meliloti. Ribozyme constructs derived from intron RmInt1 self-splice in vitro when incubated under permissive conditions, but the excised intron and ligated exons are largely replaced by unconventional products. These include a slightly shorter, 5'-end truncated 3' exon, truncated variants of the linear and lariat forms of the intron-3' exon reaction intermediate, as well as presumably circular molecules derived from the latter. Two factors explain the abundance of these products: (i) nucleotides 5-11 of the 3' exon (IBS1*) provide a better match to the EBS1 5'-exon-binding site than the authentic IBS1 sequence in the 5' exon; (ii) exon ligation is unusually inefficient, and especially so when the 5' exon is truncated close to the second (IBS2) intron-binding site. We propose that reactions at the IBS1* site play a part in the regulation of the intron ISRm2011-2 host in vivo.

Alternative Splicing↗

PubMed2006

Analysis of the exon-intron structures of fish, amphibian, bird and mammalian hatching enzyme genes, with special reference to the intron loss evolution of hatching enzyme genes in Teleostei.

Using gene cloning and in silico cloning, we analyzed the structures of hatching enzyme gene orthologs of vertebrates. Comparison led to a hypothesis that hatching enzyme genes of Japanese eel conserve an ancestral structure of the genes of fishes, amphibians, birds and mammals. However, the exon-intron structure of the genes was different from species to species in Teleostei: Japanese eel hatching enzyme genes were 9-exon-8-intron genes, and zebrafish genes were 5-exon-4-intron genes. In the present study, we further analyzed the gene structures of fishes belonging to Acanthopterygii. In the species of Teleostei we examined, diversification of hatching enzyme gene into two paralogous genes for HCE (high choriolytic enzyme) and LCE (low choriolytic enzyme) was found only in the acanthopterygian fishes such as medaka Oryzias latipes, Fundulus heteroclitus, Takifugu rubripes and Tetraodon nigroviridis. In addition, the HCE gene had no intron, while the LCE gene consisted of 8 exons and 7 introns. Phylogenetic analysis revealed that HCE and LCE genes were paralogous to each other, and diverged during the evolutionary lineage to Acanthopterygii. Analysis of gene synteny and cluster structure showed that the syntenic genes around the HCE and LCE genes were highly conserved between medaka and Teraodon, but such synteny was not found around the zebrafish hatching enzyme genes. We hypothesize that the zebrafish hatching enzyme genes were translocated from chromosome to chromosome, and lost some of their introns during evolution.

Amino Acid Sequence↗

PubMed2006

A spliceosomal intron binding protein, IBP160, links position-dependent assembly of intron-encoded box C/D snoRNP to pre-mRNA splicing.

Pre-mRNA splicing in vertebrates is molecularly linked to other processes. We previously reported that splicing is required for efficient assembly of intron-encoded box C/D small nucleolar ribonucleoprotein (snoRNP). In the spliceosomal C1 complex, snoRNP proteins efficiently assemble onto snoRNA sequences if they are located about 50 nt upstream of the intron branchpoint. Here, we identify the splicing factor responsible for coupling snoRNP assembly to intron excision. Intron binding protein (IBP) 160, a helicase-like protein previously detected in the spliceosomal C1 complex, binds the pre-mRNA in a sequence-independent manner, contacting nucleotides 33-40 upstream of the intron branch site, regardless of whether a snoRNA is present. Depletion of IBP160 abrogates snoRNP assembly in vitro. IBP160 binding directly to a snoRNA located too close to the intron branch site interferes with snoRNP assembly. Thus, IBP160 is the key factor linking snoRNP biogenesis and perhaps other postsplicing events to pre-mRNA splicing.

Animals↗

PubMed2000

The intron-containing L3 ribosomal protein gene (RPL3): sequence analysis and identification of U43 and of two novel intronic small nucleolar RNAs.

Isolation and sequencing of bovine and human intron-containing L3 ribosomal protein genes are here reported. They exhibit very similar organisation, both comprising 10 exons and nine introns. A polymorphic locus, involving a 19-bp deletion, was found in intron 6 of the human gene. The frequency of the two alleles has been estimated in 200 haploid genomes. In bovine and human genes intron sequences are rather different, except for limited regions, located in corresponding positions, which show a surprisingly high degree of identity. All these regions contain conserved features defining the box C/D class of small nucleolar RNAs. Demonstration is given that U43 small nucleolar RNA is encoded within the first intron of both bovine and human genes. Single nucleotide sequences, encoding two novel species of small nucleolar RNAs (U82, U83a and U83b), are located in introns 3, 5 and 7. Their expression has been investigated and a possible role of these molecules in 2'-O-ribose methylation of rRNAs is discussed.

Animals↗

PubMed2002

In vivo selection of better self-splicing introns in Escherichia coli: the role of the P1 extension helix of the Tetrahymena intron.

In vivo selection was used to improve the activity of the Tetrahymena pre-rRNA self-splicing intron in the context of heterologous exons. The intron was engineered into a kanamycin nucleotidyltransferase gene, with the pairing between intron bases and the 5' and 3' splice sites maintained. The initial construct failed to confer kanamycin resistance on Escherichia coli, although the pre-mRNA was active in splicing in vitro. Random mutation libraries were constructed to identify active intron variants in E. coli. All the active mutants sequenced contained mutations disrupting a base-paired region above the paired region P1 (referred to as the P1 extension region or P1ex) that involves the very 5' end of the intron. Subsequent site-directed mutagenesis confirmed that these P1ex mutations are responsible and sufficient to activate the intron splicing in E. coli. Thus, it appears that too strong of a secondary structure in the P1ex element can be inhibitory to splicing in vivo. In vitro splicing assays demonstrated that two P1ex mutant constructs splice six to eight times faster than the designed construct at 40 microM GTP concentration. The relative reaction rates of the mutant constructs compared to the original design are further increased at a lower GTP concentration. Possible mechanisms by which the disrupted P1ex structure could influence splicing rates are discussed. This study emphasizes the value of using libraries of random mutations to improve the activity of ribozymes in heterologous contexts in vivo.

Animals↗

PubMed1989

Structure-function relationships in a self-splicing group II intron: a large part of domain II of the mitochondrial intron aI5 is not essential for self-splicing.

An oligonucleotide-directed deletion of 156 nucleotides has been introduced into the yeast mitochondrial group II intron al5 (887 nt). The deletion comprises almost all of domain II, which is one of the six phylogenetically conserved structural elements of group II introns. This mutant displays reduced self-splicing activity, but results of chemical probing with dimethylsulphate suggest that sequences at the site of the deletion interfere with the normal folding of the intron. This is supported by computer analyses, which predict a number of alternative structures involving conserved intron sequences. Splicing activity could be restored by insertion of a 10-nucleotide palindromic sequence into the unique Smal site of the deletion mutant, resulting in the formation of a small stable stem-loop element at the position of domain II. These results provide a direct correlation between folding of the RNA and its activity. We conclude that at least a large part of domain II of the group II intron al5 is not required for self-splicing activity. This deletion mutant with a length of 731 nucleotides represents the smallest self-splicing group II intron so far known.

Base Sequence↗