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PubMed1998

Three different group I introns in the nuclear large subunit ribosomal DNA of the amoeboflagellate Naegleria.

We have amplified the large subunit ribosomal DNA (LSUrDNA) of the 12 described Naegleria spp. and of 34 other Naegleria lineages that might be distinct species. Two strains yielded a product that is longer than 3 kb, which is the length of the LSUrDNA of all described Naegleria spp. Sequencing data revealed that the insert in one of these strains is a group I intron without an open reading frame (ORF), while the other strain contains two different group I introns, of which the second intron has an ORF of 175 amino acids. In the latter ORF there is a conserved His-Cys box, as in the homing endonucleases present in group I introns in the small subunit ribosomal DNA (SSUrDNA) of Naegleria spp. Although the group I introns in the LSUrDNA differ in sequence, they are more related to each other than they are to the group I introns in the SSUrDNA of Naegleria spp. The three group I introns in the LSUrDNA in Naegleria are at different locations and are probably acquired by horizontal transfer, contrary to the SSUrDNA group I introns in this genus which are of ancestral origin and are transmitted vertically.

Animals↗

PubMed1998

Efficient 3'-end formation of human beta-globin mRNA in vivo requires sequences within the last intron but occurs independently of the splicing reaction.

The second intron (betaIVS-II) of the human beta-globin gene is essential for the accumulation of stable cytoplasmic mRNA and is implicated in promoting efficient 3'-end formation. This report presents quantitative comparisons between betaIVS-II mutants at physiological levels of expression from within a natural chromatin context in vivo which further defines it's function. In marked contrast to a beta-globin gene lacking a second intron, two mutants defective in splicing (small size or a splice donor mutation), still undergo essentially normal levels of 3'-end formation and in the absence of exon skipping. Therefore, 3' cleavage of beta-globin transcripts requires the presence of betaIVS-II sequences, but not the splicing reaction. The placement of betaIVS-II in the IVS-I position did not reduce the efficiency of 3' cleavage indicating that the distance between the necessary element(s) in this intron and the polyadenylation recognition site is not a crucial factor. Subsequent placement of betaIVS-I in the intron II position, reduced the efficiency of 3'-end formation to only 16% of normal. A direct replacement of intron II by the heterologous introns betaIVS-I or alpha-globin IVS-II, only partially substitute (16 and 30% respectively) for betaIVS-II. Hybrid introns show that efficient 3'-end formation is strongly enhanced by the presence of the terminal 60 nt of betaIVS-II. These data imply that the last intervening sequence of multiple intron containing genes is a principal determinant of the efficiency of 3'-end formation and may act as a post-transcriptional regulatory step in gene expression.

Animals↗

PubMed2000

YIDB: the Yeast Intron DataBase.

The Yeast Intron DataBase (YIDB) contains currently available information about all introns encoded in the nuclear and mitochondrial genomes of the yeast Saccharomyces cerevisiae. Introns are divided according to their mechanism of excision: group I and group II introns, pre-mRNA introns, tRNA introns and the HAC1 intron. Information about the host genome, the type of RNA in which they are inserted and their primary structure are provided together with references. For nuclear pre-mRNA introns, transcription frequencies, as determined by microarray experiments, have also been included. This updated database is accessible at: http://www.embl-heidelberg. de/ExternalInfo/seraphin/yidb.html

Databases, Factual↗

PubMed2000

Transcripts of the ndhH-D operon of barley plastids: possible role of unedited site III in splicing of the ndhA intron.

The plastid ndhH-D operon produces several transcripts containing ndhA sequence with and without its group II intron. After sequencing an 8125 bp fragment of barley plastid DNA including the ndhH-D operon, we investigated the editing-splicing status of transcripts in the range 1.0-7.8 kb. Reverse transcription and sequencing of RNA bands separated by electrophoresis were used to determine C-->U editing sites. Sites I, II and IV of ndhA and site V of ndhD were edited in all transcripts analysed and, probably, were edited before any splicing had taken place. In contrast, site III of ndhA (13 bp from the 5'-end base of the second exon) was not edited in transcripts containing the intron (including the 1.7 kb intermediary transcript consisting of the intron and the second exon) but was edited in all transcripts lacking the ndhA intron. Comparison of the secondary structures of the ndhA intron and intron-second exon intermediate suggests that G pairing prevents editing of site III in transcripts containing the intron and maintains the secondary structure required for splicing. Splicing of the ndhA intron releases the site III C from pairing and, probably, brings it close to cis-acting elements for editing upstream in the first exon.

Base Sequence↗

PubMed2005

Splicing of human immunodeficiency virus RNA is position-dependent suggesting sequential removal of introns from the 5' end.

Transcription of the HIV-1 genome yields a single primary transcript, which is alternatively spliced to >30 mRNAs. Productive infection depends on inefficient and regulated splicing and appears to proceed in a tight 5' to 3' order. To analyse whether sequential splicing is mediated by the quality of splice sites or by the position of an intron, we inserted the efficient beta-globin intron (BGI) into the 3' region or 5'UTR of a subgenomic expression vector or an infectious proviral plasmid. RNA analysis revealed splicing of the 3' BGI only if all upstream introns were removed, while splicing of the same intron in the 5'UTR was efficient and independent of further splicing. Furthermore, mutation of the upstream splice signal in the subgenomic vector did not eliminate the inhibition of 3' splicing, although the BGI sequence was the only intron in this case. These results suggest that downstream splicing of HIV-1 RNAs is completely dependent on prior splicing of all upstream intron(s). This hypothesis was supported by the mutation of the major 5' splice site in the HIV-1 genome, which completely abolished all splicing. It appears likely that the tight order of splicing is important for HIV-1 replication, which requires the stable production of intron containing RNAs, while splicing of 3' introns on incompletely spliced RNAs would be likely to render them subject to nonsense-mediated decay.

5' Untranslated Regions↗

PubMed2005

The excess of 5' introns in eukaryotic genomes.

In this work, 21 completely sequenced eukaryotic genomes were analyzed using an intragene comparison approach. We found that all of these genomes show a significant 5'-biased distribution of introns of protein-coding genes. Our findings are different from previous studies based on the intergene method, where introns are biased towards the 5' end of genes only in intron-poor genomes, but are evenly distributed in intron-rich genomes. In addition, by analyzing the patterns of intron distribution of a set of well-compiled housekeeping genes from human and their respective orthologs identified by a bidirectional best BLAST hit method from the other genomes, we found that the trend of 5'-biased intron positions of the set of housekeeping genes for each genome is much more skewed than that of all genes of the same genome, and rarely if any of the housekeeping genes examined have an extremely 3'-biased position distribution in which all introns of a gene are located only at the 3' portion of the gene. The most parsimonious explanation for our findings may be the model in which intron loss is caused by homologous recombination between the genomic copy of a gene and a reverse transcriptase product of a spliced mRNA.

Animals↗

PubMed2006

A novel multifunctional factor involved in trans-splicing of chloroplast introns in Chlamydomonas.

In the chloroplast of Chlamydomonas reinhardtii, psaA mRNA is spliced in trans from three separate precursors which assemble to form two group II introns. A fourth transcript, tscA, completes the structure of the first intron. Of the fourteen nucleus-encoded factors involved in psaA splicing, only two are required for splicing of both introns. We cloned and characterized the first of these more general factors, Raa1. Consistently with its role in psaA splicing, Raa1 is imported in the chloroplast where it is found in a membrane fraction and is part of a large ribonucleoprotein complex. One mutant, raa1-L137H, is defective for splicing of both introns, but another allelic mutant, raa1-314B, still expresses the 3' part of the Raa1 gene and is deficient only in splicing of intron 2. This observation and a deletion analysis indicate the presence of two domains in Raa1. The C-terminal domain is necessary and sufficient for processing of tscA RNA and splicing of the first intron, while the central domain is essential for splicing of the second intron. The combination of these two functional domains in Raa1 suggests that this new factor may coordinate trans-splicing of the two introns to improve the efficiency of psaA maturation.

Algal Proteins↗

PubMed1999

Structural characteristics and possible horizontal transfer of group I introns between closely related plant pathogenic fungi.

We have characterized structural features and the distribution pattern of nuclear group I introns found in ribosomal DNA (rDNA) of closely related plant pathogenic fungi of the family Sclerotiniaceae. Sixteen introns, at two distinct positions in the small-subunit (SSU) and large-subunit (LSU) rDNA, were sequenced and analyzed among the 29 taxa included in the initial screening. Genera found to contain introns were Botrytis, Dumontinia, Encoelia, Grovesinia, Myriosclerotinia, and Sclerotinia. Secondary-structure analyses of the group I introns concluded that all belong to the common IC1 subclass. Interestingly, the SSU rDNA intron from Myriosclerotinia caricisampullacea contains an insertion-like sequence extension which may be a relic of an open reading frame. Incongruent branching patterns of intron-based and rDNA-based (internal transcribed spacer) phylogenetic trees suggest that the fungal host genomes and the group I introns do not share a common evolutionary history. A model to explain how horizontal intron transfers may have occurred among the closely related fungal taxa is proposed.

Amino Acid Sequence↗

PubMed1993

Evidence for independent acquisition of group I introns in green algae.

We report the occurrence of a group I intron, 452 nucleotides in length, in the nuclear small-subunit ribosomal gene of the benthic seaweed Urospora penicilliformis, a member of the green algal class Ulvophyceae. Group I introns have been reported in fungi, myxomycetes, the ciliate genus Tetrahymena, and recently in five unicellular chlorophycean algae. The sequence of the conserved core of the U. penicilliformis group I intron was aligned with that of 15 other introns of this type, and the evolutionary relationships among these introns were examined. Comparison of the "intron" tree with phylogenetic hypotheses of the intron-harboring organisms shows that nuclear group I introns in green algae have arisen several times and are not lineage specific.

Animals↗

PubMed1995

Evidence for insertion of a new intron into an Mhc gene of perch-like fish.

The evolution of the major histocompatibility complex (Mhc) has been studied to understand the origin of the immune system, of which it constitutes an essential part. In the present study, the Mhc is used to shed light on questions regarding the origin of introns and the phylogeny of fishes. The organization of the coding (exon) and non-coding (intron) regions of both class I and class II major histocompatibility complex (Mhc) genes is highly conserved in all vertebrate classes; the only variation observed until now is in the number of exons encoding the membrane-anchoring part. Moreover, there is a good correspondence between the exon-intron organization at the DNA level and the division into structurally and functionally defined domains at the protein level. Here we describe the first major exception to this uniformity. The immunoglobulin-like domain of the class II beta-chains in perch-like fishes (Percomorpha) is not encoded in one exon, as it is in all other vertebrates studied thus far, but in two exons. The length of the extra intron varies from gene to gene and from species to species, but is generally less than 200 base pairs (b.p.). Only one of the sequenced introns is about 500 b.p. long. In some of the genes, the intron contains a hexamer repeat. The repeat is present in the transcript at the site at which the intron interrupts exon 3 in the genomic DNA. The intron may therefore have arisen by repeated tandem duplication of this sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1998

Intron polymorphism in small subunit rDNA of Nectria galligena.

PCR amplification of the small subunit (SSU) rDNA gene of 40 isolates of Nectria galligena revealed four length polymorphisms. PCR-RFLP analysis of the SSU rDNA gene divided the isolates into four categories similar, but not identical, to categories identified by Southern-RFLP analysis. Nucleotide sequence analysis revealed that isolates in three of the four SSU rDNA (18S) categories possess an intron of 363 bp, 1185 bp or 1423 bp at the NS 7 priming site. Isolates in the fourth category do not possess an intron. The nucleotide sequences of these introns did not contain the core elements characteristic of typical group I introns, nor did they exhibit a group I intron secondary structure. Homology between the introns indicates a common lineage, all three possibly having come from a larger intron and having been formed by subsequent deletions. PCR primers upstream of the SSU rDNA intron region and from within the internal transcribed spacer 1 region amplify a product specific to N. galligena, which will confirm the identity of the pathogen and reveal its 18S category in a single reaction.

DNA, Fungal↗

PubMed1995

Exonucleolytic processing of small nucleolar RNAs from pre-mRNA introns.

Many small nucleolar RNAs (snoRNAs) in vertebrates are encoded within introns of protein genes. We have reported previously that two isoforms of human U17 snoRNA are encoded in introns of the cell-cycle regulatory gene, RCC1. We have now investigated the mechanism of processing of U17 RNAs and of another intron-encoded snoRNA, U19. Experiments in which the processing of intronic RNA substrates was tested in HeLa cell extracts suggest that exonucleases rather than endonucleases are involved in the excision of U17 and U19 RNAs: (1) Cutoff products that would be expected from endonucleolytic cleavages were not detected; (2) capping or circularization of substrates inhibited formation of snoRNAs; and (3) U17 RNA was faithfully processed from a substrate carrying unrelated flanking sequences. To study in vivo processing the coding regions of snoRNAs were inserted into intron 2 of the human beta-globin gene. Expression of resulting pre-mRNAs in simian COS cells resulted in formation of correctly processed snoRNAs and of the spliced globin mRNA, demonstrating that snoRNAs can be excised from a nonhost intron and that their sequences contain all the signals essential for accurate processing. When the U17 sequence was placed in a beta-globin exon, no formation of U17 RNA took place, and when two U17 RNA-coding regions were placed in a single intron, doublet U17 RNA molecules accumulated. The results support a model according to which 5'-->3' and 3'-->5' exonucleases are involved in maturation of U17 and U19 RNAs and that excised and debranched introns are the substrates of the processing reaction.

Base Sequence↗

PubMed2007

Widespread mRNA polyadenylation events in introns indicate dynamic interplay between polyadenylation and splicing.

mRNA polyadenylation and pre-mRNA splicing are two essential steps for the maturation of most human mRNAs. Studies have shown that some genes generate mRNA variants involving both alternative polyadenylation and alternative splicing. Polyadenylation in introns can lead to conversion of an internal exon to a 3' terminal exon, which is termed composite terminal exon, or usage of a 3' terminal exon that is otherwise skipped, which is termed skipped terminal exon. Using cDNA/EST and genome sequences, we identified polyadenylation sites in introns for all currently known human genes. We found that approximately 20% human genes have at least one intronic polyadenylation event that can potentially lead to mRNA variants, most of which encode different protein products. The conservation of human intronic poly(A) sites in mouse and rat genomes is lower than that of poly(A) sites in 3'-most exons. Quantitative analysis of a number of mRNA variants generated by intronic poly(A) sites suggests that the intronic polyadenylation activity can vary under different cellular conditions for most genes. Furthermore, we found that weak 5' splice site and large intron size are the determining factors controlling the usage of composite terminal exon poly(A) sites, whereas skipped terminal exon poly(A) sites tend to be associated with strong polyadenylation signals. Thus, our data indicate that dynamic interplay between polyadenylation and splicing leads to widespread polyadenylation in introns and contributes to the complexity of transcriptome in the cell.

Animals↗

PubMed2003

Comparative analysis of vertebrate dystrophin loci indicate intron gigantism as a common feature.

The human DMD gene is the largest known to date, spanning > 2000 kb on the X chromosome. The gene size is mainly accounted for by huge intronic regions. We sequenced 190 kb of Fugu rubripes (pufferfish) genomic DNA corresponding to the complete dystrophin gene (FrDMD) and provide the first report of gene structure and sequence comparison among dystrophin genomic sequences from different vertebrate organisms. Almost all intron positions and phases are conserved between FrDMD and its mammalian counterparts, and the predicted protein product of the Fugu gene displays 55% identity and 71% similarity to human dystrophin. In analogy to the human gene, FrDMD presents several-fold longer than average intronic regions. Analysis of intron sequences of the human and murine genes revealed that they are extremely conserved in size and that a similar fraction of total intron length is represented by repetitive elements; moreover, our data indicate that intron expansion through repeat accumulation in the two orthologs is the result of independent insertional events. The hypothesis that intron length might be functionally relevant to the DMD gene regulation is proposed and substantiated by the finding that dystrophin intron gigantism is common to the three vertebrate genes.

Amino Acid Sequence↗

PubMed2006

Arabidopsis orthologs of maize chloroplast splicing factors promote splicing of orthologous and species-specific group II introns.

Chloroplast genomes in plants and green algae contain numerous group II introns, large ribozymes that splice via the same chemical steps as spliceosome-mediated splicing in the nucleus. Most chloroplast group II introns are degenerate, requiring interaction with nucleus-encoded proteins to splice in vivo. Genetic approaches in maize (Zea mays) and Chlamydomonas reinhardtii have elucidated distinct sets of proteins that assemble with chloroplast group II introns and facilitate splicing. Little information is available, however, concerning these processes in Arabidopsis (Arabidopsis thaliana). To determine whether the paucity of data concerning chloroplast splicing factors in Arabidopsis reflects a fundamental difference between protein-facilitated group II splicing in monocot and dicot plants, we examined the mutant phenotypes associated with T-DNA insertions in Arabidopsis genes encoding orthologs of the maize chloroplast splicing factors CRS1, CAF1, and CAF2 (AtCRS1, AtCAF1, and AtCAF2). We show that the splicing functions and intron specificities of these proteins are largely conserved between maize and Arabidopsis, indicating that these proteins were recruited to promote the splicing of plastid group II introns prior to the divergence of monocot and dicot plants. We show further that AtCAF1 promotes the splicing of two group II introns, rpoC1 and clpP-intron 1, that are found in Arabidopsis but not in maize; AtCAF1 is the first splicing factor described for these introns. Finally, we show that a strong AtCAF2 allele conditions an embryo-lethal phenotype, adding to the body of data suggesting that cell viability is more sensitive to the loss of plastid translation in Arabidopsis than in maize.

Arabidopsis↗

PubMed2004

Determinants of plant U12-dependent intron splicing efficiency.

Factors affecting splicing of plant U12-dependent introns have been examined by extensive mutational analyses in an in vivo tobacco (Nicotiana tabacum) protoplast system using introns from three different Arabidopsis thaliana genes: CBP20, GSH2, and LD. The results provide evidence that splicing efficiency of plant U12 introns depends on a combination of factors, including UA content, exon bridging interactions between the U12 intron and flanking U2-dependent introns, and exon splicing enhancer sequences (ESEs). Unexpectedly, all three plant U12 introns required an adenosine at the upstream purine position in the branchpoint consensus UCCUURAUY. The exon upstream of the LD U12 intron is a major determinant of its higher level of splicing efficiency and potentially contains two ESE regions. These results suggest that in plants, U12 introns represent a level at which expression of their host genes can be regulated.

Arabidopsis↗

PubMed2005

HLA-DQB1 sequencing-based typing using newly identified conserved nucleotide sequences in introns 1 and 2.

Sequencing-based typing (SBT) human leukocyte antigen (HLA) class I and II genes should examine entire exon sequences where polymorphisms lie. Primers for the amplification of complete exons therefore anneal in introns and their design relies on accurate intron sequences being available. We decided to develop a SBT method for HLA-DQB1 using amplification primers which anneal in introns 1 and 2, yet the amount of intron sequence data previously available in databases was sparse. Therefore, we undertook a systematic sequencing of introns 1 and 2 using DNA from cell lines homozygous for DQB1. This study confirmed an earlier report that the non-coding regions of this gene are the most polymorphic seen in the human genome. Intron sequences within an allele group were largely identical, the exceptions being DQB1*0301 differing from other DQB1*03 allele groups and DQB1*0601 differing from all other DQB1*06 alleles. A retroviral Alu element, related to the AluYa5a2 subfamily, was identified uniquely inserted in intron 2 of DQB1*02 alleles. For the typing approach, six amplification primers were designed based on conserved allele group sequences covering all of the HLA DQB antigens, and two sequencing primers were also designed which anneal in intron 2. This method has proved to be very robust and has been used as part of a referral DNA sequencing service for a number of years.

Alleles↗

PubMedDate not supplied

Dynamics and function of intron sequences of the wingless gene during the evolution of the Drosophila genus.

To understand the function and evolution of genes with complex patterns of expression, such as the Drosophila wingless gene, it is essential to know how their transcription is regulated. However, extracting the relevant regulatory information from a genome is still a complex task. We used a combination of comparative genomics and functional approaches to identify putative regulatory sequences in two introns (1 and 3) of the wingless gene and to infer their evolution. Comparison of the sequences obtained from several Drosophila species revealed colinear and well-conserved sequence blocks in both introns. Drosophila willistoni showed a rate of evolution, in both introns, faster than expected from its phylogenetic position. Intron 3 appeared to be composed of two separate modules, one of them lost in the willistoni group. We tested whether sequence conservation in noncoding regions is a reliable indicator of regulatory function and, if this function is conserved, by analyzing D. melanogaster transgenic reporter lines harboring intron 3 sequences from D. melanogaster (Sophophora subgenus) and the species from the Drosophila subgenus presenting the most divergent sequence, D. americana. The analysis indicated that intron 3 contains pupal enhancers conserved during the evolution of the genus, despite the fact that only 30% of the D. melanogaster intron 3 sequences lie in conserved blocks. Additional analysis of D. melanogaster transgenic reporter lines harboring intron 3 sequences from D. willistoni revealed the absence of an abdomen-specific expression pattern, probably due to the above-mentioned loss of a regulatory module in this species.

Animals↗