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PubMed1998

T-->G or T-->A mutation introduced in the branchpoint consensus sequence of intron 4 of lecithin:cholesterol acyltransferase (LCAT) gene: intron retention causing LCAT deficiency.

Previous mutations associated with lecithin:cholesteryl acyltransferase (LCAT) deficiency syndromes have been identified in the coding regions of the LCAT gene. However, recently, an intron mutation was found in a family in which three sisters presented with fish-eye disease (FED). The probands were shown to be heterozygotes for a mutation in intron 4. The respective T-->C nucleotide substitution, 22 bases upstream of the 3'-splice site, causes a null allele as the result of complete intron retention. Since the natural mutation occurs in a putative branchpoint consensus sequence of the intron, it was hypothesized that the point mutation may disrupt the splicing of the pre-mRNA. To further study the functional significance of the above thymine residue in the branchpoint sequence, we introduced other nucleotides at this position, i.e., LCAT Int-4 MUT-1 (T-->G) and LCAT Int-4 MUT-2 (T-->A). After stable transfection of the mutated pNUT-LCAT minigenes into BHK cells, we could detect neither LCAT activity nor LCAT protein in the culture medium of the pNUT-LCAT Int-4 MUT-1 and pNUT-LCAT Int-4 MUT-2 cell lines, as was previously described for the natural mutation. To determine the effects of the introduced mutations on pre-mRNA splicing, total RNA from transfected BHK cells was used for RT-PCR analysis. All BHK cell lines were shown to transcribe the integrated LCAT minigenes. However, the sizes of these LCAT messengers indicated that intron 4 was retained in the pNUT-LCAT Int-4 MUT-1 and pNUT-LCAT Int-4 MUT-2 cell lines. Subsequent sequence analysis of the RT-PCR products demonstrated that the unspliced intronic sequences contained the introduced mutations. In conclusion, the observed retention of intron 4 of the LCAT gene is the result of the specific loss of a thymine residue two bases upstream of the branchpoint adenosine residue in the putative branchpoint consensus sequence. The results confirm that a single base change in the branchpoint consensus sequence of an intron can cause human disease although this sequence is poorly conserved in mammals.

Animals↗

PubMed1996

A novel group I intron in Candida dubliniensis is homologous to a Candida albicans intron.

In the present study, we determined the sequence of group I self-splicing introns found in the large ribosomal RNA subunit of Candida albicans, Candida stellatoidea and the recently-described species Candida dubliniensis. It was found that both the intron and ribosomal RNA nucleotide sequences are almost perfectly identical between different C. albicans strains as well as between C. albicans and C. stellatoidea strains. Comparisons of ribosomal RNA sequences suggest that local isolates of atypical C. albicans from individuals infected with human immunodeficiency virus can be assigned to the C. dubliniensis species. C. dubliniensis strains also harbor a group I intron in their ribosomal RNA, as observed in about 40% of C. albicans strains and all C. stellatoidea strains. This novel C. dubliniensis group I intron is identical to the C. albicans and C. stellatoidea intron, except for two widely divergent stem-loop regions. Despite these differences, the C. dubliniensis intron possesses self-splicing ability in an in vitro assay. Taken together, these data support the idea that C. albicans and C. stellatoidea should be joined together as variants of the same species while C. dubliniensis is a distinct but closely related microorganism. To our knowledge, the C. albicans and C. dubliniensis introns are the first example of a pair of homologous group I introns differing only by the presence of apparently facultative sequences in some stem-loops suspected to be involved in stabilization of tertiary structure.

Base Sequence↗

PubMed1995

Conservation of the sizes of 53 introns and over 100 intronic sequences for the binding of common transcription factors in the human and mouse genes for type II procollagen (COL2A1).

Over 11,000 bp of previously undefined sequences of the human COL2A1 gene were defined. The results made it possible to compare the intron structures of a highly complex gene from man and mouse. Surprisingly, the sizes of the 53 introns of the two genes were highly conserved with a mean difference of 13%. After alignment of the sequences, 69% of the intron sequences were identical. The introns contained consensus sequences for the binding of over 100 different transcription factors that were conserved in the introns of the two genes. The first intron of the gene contained 80 conserved consensus sequences and the remaining 52 introns of the gene contained 106 conserved sequences for the binding of transcription factors. The 5'-end of intron 2 in both genes had a potential for forming a stem loop in RNA transcripts.

Animals↗

PubMed1988

Plant intron sequences: evidence for distinct groups of introns.

In vivo and in vitro RNA splicing experiments have demonstrated that the intron splicing machineries are not interchangeable in all organisms. These differences have prevented the efficient in vivo expression of monocot genes containing introns in dicot plants and the in vitro excision of some plant introns in HeLa cell in vitro splicing extracts. We have analyzed plant introns for sequence differences which potentially account for the functional splicing differences. Three classes of plant introns can be differentiated by the purine or pyrimidine-richness of sequences upstream from the 3' splice site. The frequency of these three types of introns in monocots and dicots varies significantly. The degree of variability in the 5' and 3' intron boundaries is evaluated for each of these classes in monocots and dicots. The 5' splice site consensus sequences developed for the monocot and dicot introns differ in their ability to base pair with conserved nucleotides present at the 5' end of many U1 snRNAs.

Base Sequence↗

PubMed1999

Evolution of the mitochondrial rps3 intron in perennial and annual angiosperms and homology to nad5 intron 1.

The plant mitochondrial rps3 intron was analyzed for substitution and indel rate variation among 15 monocot and dicot angiosperms from 10 genera, including perennial and annual taxa. Overall, the intron sequence was very conserved among angiosperms. Based on length polymorphism, 10 different alleles were identified among the 10 genera. These allelic differences were mainly attributable to large indels. An insertion of 133 nucleotides, observed in the Alnus intron was partially or completely absent in the other lineages of the family Betulaceae. This insertion was located within domain IV of the secondary-structure model of this group IIA intron. A mobile element of 47 nucleotides that showed homology to sequences located in rice rps3 intron and in intergenic plant mitochondrial genomes was found within this insertion. Both substitution and indel rates were low among the Betulaceae sequences, but substitution rates were increasingly larger than indel rates in comparisons involving more distantly related taxa. From a secondary-structure model, regions involved in helical structures were shown to be well preserved from indels as compared to substitutions, but compensatory changes were not observed among the angiosperm sequences analyzed. Using approximate divergence times based on the fossil record, substitution and indel rate heterogeneity was observed between different pairs of annual and perennial taxa. In particular, the annual petunia and primrose evolved more than 15 and 10 times faster, for substitution and indel rates respectively, than the perennial birch and alder. This is the first demonstration of an evolutionary rate difference between perennial and annual forms in noncoding DNA, lending support to neutral causes such as the generation time, population size, and speciation rate effects to explain such rate heterogeneity. Surprisingly, the sequence from the rps3 intron had a high identity with the sequence of intron 1 from the angiosperm mitochondrial nad5 gene, suggesting a common origin of these two group IIA introns.

Base Sequence↗

PubMed2004

Repeat-type distribution in trnL intron does not correspond with species phylogeny: comparison of the genetic markers 16S rRNA and trnL intron in heterocystous cyanobacteria.

tRNA(Leu) UAA (trnL) intron sequences are used as genetic markers for differentiating cyanobacteria and for constructing phylogenies, since the introns are thought to be more variable among close relatives than is the 16S rRNA gene, the conventional phylogenetic marker. The evolution of trnL intron sequences and their utility as a phylogenetic marker were analysed among heterocystous cyanobacteria with maximum-parsimony, maximum-likelihood and Bayesian inference by comparing their evolutionary information to that of the 16S rRNA gene. Trees inferred from the 16S rRNA gene and the distribution of two repeat classes in the P6b stem-loop of the trnL intron were in clear conflict. The results show that, while similar heptanucleotide repeat classes I and II in the P6b stem-loop of the trnL intron could be found among distant relatives, some close relatives harboured different repeat classes with a high sequence difference. Moreover, heptanucleotide repeat class II and other sequences from the P6b stem-loop of the trnL intron interrupted several other intergenic regions in the genomes of heterocystous cyanobacteria. Cluster analyses based on conserved intron sequences without loops P6b, P9 and parts of P5 corresponded in most clades to the 16S rRNA gene phylogeny, although the relationships were not resolved well, according to low bootstrap support. Thus, the hypervariable loop sequences of the trnL intron, especially the P6b stem-loop, cannot be used for phylogenetic analysis and conclusions cannot be drawn about species relationships on the basis of these elements. Evolutionary scenarios are discussed considering the origin of the repeats.

Base Sequence↗

PubMed1994

Intron definition in splicing of small Drosophila introns.

Approximately half of the introns in Drosophila melanogaster are too small to function in a vertebrate and often lack the pyrimidine tract associated with vertebrate 3' splice sites. Here, we report the splicing and spliceosome assembly properties of two such introns: one with a pyrimidine-poor 3' splice site and one with a pyrimidine-rich 3' splice site. The pyrimidine-poor intron was absolutely dependent on its small size for in vivo and in vitro splicing and assembly. As such, it had properties reminiscent of those of yeast introns. The pyrimidine-rich intron had properties intermediate between those of yeasts and vertebrates. This 3' splice site directed assembly of ATP-dependent complexes when present as either an intron or exon and supported low levels of in vivo splicing of a moderate-length intron. We propose that splice sites can be recognized as pairs across either exons or introns, depending on which distance is shorter, and that a pyrimidine-rich region upstream of the 3' splice site facilitates the exon mode.

Animals↗

PubMed2003

The ability to form full-length intron RNA circles is a general property of nuclear group I introns.

In addition to splicing, group I intron RNA is capable of an alternative two-step processing pathway that results in the formation of full-length intron circular RNA. The circularization pathway is initiated by hydrolytic cleavage at the 3' splice site and followed by a transesterification reaction in which the intron terminal guanosine attacks the 5' splice site presented in a structure analogous to that of the first step of splicing. The products of the reactions are full-length circular intron and unligated exons. For this reason, the circularization reaction is to the benefit of the intron at the expense of the host. The circularization pathway has distinct structural requirements that differ from those of splicing and appears to be specifically suppressed in vivo. The ability to form full-length circles is found in all types of nuclear group I introns, including those from the Tetrahymena ribosomal DNA. The biological function of the full-length circles is not known, but the fact that the circles contain the entire genetic information of the intron suggests a role in intron mobility.

Base Sequence↗

PubMed1994

Self-splicing group I intron in cyanobacterial initiator methionine tRNA: evidence for lateral transfer of introns in bacteria.

A group I self-splicing intron has been found in the anticodon loop of tRNA(fMet) genes in three cyanobacterial genera: Dermocarpa, Scytonema and Synechocystis; it is absent in nine others. The Synechocystis intron is also interrupted by an open reading frame (ORF) of 150 codons. Of these three bacteria, only Scytonema also contains the group I intron that has previously been reported in tRNA(Leu) (UAA) genes in both cyanobacteria and chloroplasts. The presence of an ORF in the tRNA(fMet) intron, the sporadic distribution of the intron among cyanobacteria and the lack of correlation between relatedness of the intron sequences and the bacteria in which they reside, are all consistent with recent introduction of this intron by lateral transfer.

Amino Acid Sequence↗

PubMed2000

The distribution of group I introns in lichen algae suggests that lichenization facilitates intron lateral transfer.

The nuclear-encoded small subunit ribosomal DNA gene of many lichen-forming green algae in the genus Trebouxia contains a group I intron at Escherichia coli genic position 1512. We studied the evolutionary history of the 1512 intron in Trebouxia spp. (Trebouxiophyceae) by analyzing intron and "host" cell phylogenies. The host trees were constructed by comparing internal transcribed spacer regions of rDNA. Maximum-likelihood, maximum-parsimony, and distance analyses suggest that the 1512 intron was present in the common ancestor of the green algal classes Trebouxiophyceae, Chlorophyceae, and Ulvophyceae. The 1512 intron, however, was laterally transferred at least three times among later-diverging Trebouxia spp. that form lichen partnerships. Intron secondary structure analyses are consistent with this result. Our results support the hypothesis that lichenization may facilitate 1512 group I intron lateral transfer through the close cell-to-cell contact that occurs between the lichen algal and fungal symbionts in the developing lichen thallus.

Alternative Splicing↗

PubMed1997

Self-splicing activity of the mitochondrial group-I introns from Aspergillus nidulans and related introns from other species.

The mitochondrial genome of Aspergillus nidulans contains several group-I introns. Each one has been assayed for its ability to self-splice in vitro in the absence of proteins. The intron from the apocytochrome b gene is unusual among subgroup IB4 introns in being able to self-splice, unlike a similar intron from Saccharomyces cerevisiae. The first intron in the cytochrome oxidase subunit-1 gene self-splices but only correctly completes the first step of splicing; cryptic 3' splice-sites are recognized instead and these are also used at a low frequency in vivo. The highly homologous intron from Podospora anserina completes both steps in vitro. The remaining introns do not self-splice. The correlation between subgroup category, the likely presence of specific tertiary interactions, and self-splicing activity is discussed.

Apoproteins↗

PubMed1989

A latent intron-encoded maturase is also an endonuclease needed for intron mobility.

Some yeast mitochondrial introns encode proteins that promote either splicing (maturases) or intron propagation via gene conversion (the fit1 endonuclease). We surveyed introns in the coxl gene for their ability to engage in gene conversion and found that the group I intron, al4 alpha, was efficiently transmitted to genes lacking it. An endonucleolytic cleavage is detectable in recipient DNA molecules near the site of intron insertion in vivo and in vitro. Conversion is dependent on an intact al4 alpha open reading frame. This intron product is a latent maturase, but these data show that it is also a potent endonuclease involved in recombination. Dual function proteins that cleave DNA and facilitate RNA splicing may have played a pivotal role in the propagation and tolerance of introns.

Base Sequence↗

PubMed1998

Mosaic structure of the cox2 gene in the petite negative yeast Schizosaccharomyces pombe: a group II intron is inserted at the same location as the otherwise unrelated group II introns in the mitochondria of higher plants.

In contrast to homologous genes in other fungal mitochondrial genomes, the gene encoding subunit 2 of cytochrome oxidase (cox2) in several Schizosaccharomyces pombe strains contains a large group II intron. Its 2436 nucleotides can be folded into a typical group II intron secondary structure, possessing all the expected sequence motifs for subgroup IIA1 (Michel et al., 1989). This intron is remarkable for the following reasons: (i) Five nucleotide changes were observed compared with the continuous form of the cox2 gene in the reference strain 50 at the 3'-exon sequence, but not in the 5'-exon. (ii) One of these changes occurred at the splice point leading to a serine instead of a threonine residue in the deduced cox2 polypeptide. In all cases, the alterations resulted in the replacement of more frequently used codons by rare ones. (iii) Although the intron is able to undergo splicing, the sequence motifs thought to be necessary for interaction between the 5'-exon and the intron during the splicing process (the EBS1/IBS1 as well as the EBS2/IBS2 pairings) are unusual. (iv) The intron is inserted at the same location in the cox2 gene as the otherwise unrelated intron from higher plants.

Alleles↗

PubMed1997

Terminal intron dinucleotide sequences do not distinguish between U2- and U12-dependent introns.

Two types of eukaryotic nuclear introns are known: the common U2-dependent class with /GU and AG/ terminal intron dinucleotides, and the rare U12-dependent class with /AU and AC/ termini. Here we show that the U12-dependent splicing system can splice introns with /GU and AG/ termini and that such introns occur naturally. Further, U2-dependent introns with /AU and AC/termini also occur naturally and are evolutionarily conserved. Thus, the sequence of the terminal dinucleotides does not determine which spliceosomal system removes an intron. Rather, the four classes of introns described here can be sorted into two mechanistic classes (U2- or U12-dependent) by inspection of the complete set of conserved splice site sequences.

Animals↗

PubMed2006

Smoke without fire: most reported cases of intron gain in nematodes instead reflect intron losses.

Identification of recently gained spliceosomal introns would provide crucial evidence in the continuing debate concerning the age and evolutionary significance of introns. A previously published genomic analysis reported to have identified 122 introns that had been gained since the divergence of the nematodes Caenorhabidits elegans and Caenorhabditis briggsae approximately 100 MYA. However, using newly available genomic sequence from additional Caenorhabditis species, we show that 74% (60/81) of the reported gains in C. elegans are present in a C. briggsae relative. This pattern indicates that these introns represent losses in C. briggsae, not gains in C. elegans. In addition, 61% (25/41) of the reported gains in C. briggsae are present in the more distant C. briggsae relative, in a pattern suggesting that additional reported gains in C. elegans and/or C. briggsae may in fact represent unrecognized losses. These results underscore the dominance of intron loss over intron gain in recent eukaryotic evolution, the pitfalls associated with parsimony in inferring intron gains, and the importance of genomic sequencing of clusters of closely related species for drawing accurate inferences about genome evolution.

Amino Acid Sequence↗

PubMed1994

Nucleolar introns from Physarum flavicomum contain insertion elements that may explain how mobile group I introns gained their open reading frames.

Comparison of two group I intron sequences in the nucleolar genome of the myxomycete Physarum flavicomum to their homologs in the closely related Physarum polycephalum revealed insertion-like elements. One of the insertion-like elements consists of two repetitive sequence motifs of 11 and 101 bp in five and three copies, respectively. The smaller motif, which flanks the larger, resembles a target duplication and indicates a relationship to transposons or retroelements. The insertion-like elements are found in the peripheral loops of the RNA structure; the positions occupied by the ORFs of mobile nucleolar group I introns. The P. flavicomum introns are 1184 and 637 bp in size, located in the large subunit ribosomal RNA gene, and can be folded into group I intron structures at the RNA level. However, the intron 2s from both P. flavicomum and P. polycephalum contain an unusual core region that lacks the P8 segment. None of the introns are able to self-splice in vitro. Southern analysis of different isolates indicates that the introns are not optional in myxomycetes.

Animals↗

PubMed2003

The DIVa maturase binding site in the yeast group II intron aI2 is essential for intron homing but not for in vivo splicing.

Splicing of the Saccharomyces cerevisiae mitochondrial DNA group II intron aI2 depends on the intron-encoded 62-kDa reverse transcriptase-maturase protein (p62). In wild-type strains, p62 remains associated with the excised intron lariat RNA in ribonucleoprotein (RNP) particles that are essential for intron homing. Studies of a bacterial group II intron showed that the DIVa substructure of intron domain IV is a high-affinity binding site for its maturase. Here we first present in vitro evidence extending that conclusion to aI2. Then, experiments with aI2 DIVa mutant strains show that the binding of p62 to DIVa is not essential for aI2 splicing in vivo but is essential for homing. Because aI2 splicing in the DIVa mutant strains remains maturase dependent, splicing must rely on other RNA-protein contacts. The p62 that accumulates in the mutant strains has reverse transcriptase activity, but fractionation experiments at high and low salt concentrations show that it associates more weakly than the wild-type protein with endogenous mitochondrial RNAs, and that phenotype probably explains the homing defect. Replacing the DIVa of aI2 with that of the closely related intron aI1 improves in vivo splicing but not homing, indicating that DIVa contributes to the specificity of the maturase-RNA interaction needed for homing.

Base Sequence↗

PubMed2005

A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing.

Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N- and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.

Aspergillus nidulans↗