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PubMed2005

Rates of intron loss and gain: implications for early eukaryotic evolution.

We study the intron-exon structures of 684 groups of orthologs from seven diverse eukaryotic genomes and provide maximum likelihood estimates for rates and numbers of intron losses and gains in these same genes for a variety of lineages. Rates of intron loss vary from approximately 2 x 10(-9) to 2 x 10(-10) per year. Rates of gain vary from 6 x 10(-13) to 4 x 10(-12) per possible intron insertion site per year. There is an inverse correspondence between rates of intron loss and gain, leading to a 20-fold variation among lineages in the ratio of the rates of the two processes. The observed rates of intron gain are insufficient to explain the large number of introns estimated to have been present in the plant-animal ancestor, suggesting that introns present in early eukaryotes may have been created by a fundamentally different process than more recently gained introns.

Animals↗

PubMed1999

Late changes in spliceosomal introns define clades in vertebrate evolution.

The evolutionary origin of spliceosomal introns has been the subject of much controversy. Introns are proposed to have been both lost and gained during evolution. If the gain or loss of introns are unique events in evolution, they can serve as markers for phylogenetic analysis. We have made an extensive survey of the phylogenetic distribution of seven spliceosomal introns that are present in Fugu genes, but not in their mammalian homologues; we show that these introns were acquired by actinopterygian (ray-finned) fishes at various stages of evolution. We have also investigated the intron pattern of the rhodopsin gene in fishes, and show that the four introns found in the ancestral chordate rhodopsin gene were simultaneously lost in a common ancestor of ray-finned fishes. These changes in introns serve as excellent markers for phylogenetic analysis because they reliably define clades. Our intron-based cladogram establishes the difficult-to-ascertain phylogenetic relationships of some ray-finned fishes. For example, it shows that bichirs (Polypterus) are the sister group of all other extant ray-finned fishes.

Amino Acid Sequence↗

PubMed1995

Role of intron I in expression of the human factor IX gene.

The first intron (intron I) of the human factor IX gene, which has been previously suggested of having an expression-augmenting activity, was systematically studied for its potential enhancer activity. When tested with the chloramphenicol acetyltransferase expression vector with a minimal factor IX promoter, subregions of intron I showed only marginal enhancing activities (1.7-1.9-fold enhancement at the highest). Smaller subregions encompassing nucleotides 5660-6350 of the intron sequence even showed some weak negative regulatory activities (approximately 50% suppression at the highest), while a cytomegalovirus enhancer sequence, which was used as the positive control, had a 7-fold enhancement. A set of three factor IX minigene expression vectors with the same factor IX promoter were then constructed: p-416FIXc which contained the factor IX cDNA, p-416FIXm1 which contained the factor IX cDNA with a largely truncated intron I, and p-416FIXm2 which contained the factor IX cDNA with the intron I sequence further truncated. The p-416FIXm1 and p-416FIXm2 constructs showed 7-9-fold higher expression activities than p-416FIXc. The elevated factor IX antigen levels agreed well with the grossly elevated factor IX clotting activity and mRNA levels. These results indicate that the expression enhancing activity of intron I is not due to specific enhancer elements present in the intron subsequences, but is due to functional splicing sequences present in the precursor mRNAs produced from the minigene constructs containing intron I. By being efficiently assembled into spliceosome complexes, transcripts with splicing sequences may be better protected in the nucleus from random degradations than those without such sequences.

Animals↗

PubMed1995

Multiple splicing signals control alternative intron retention of bovine growth hormone pre-mRNA.

A fraction of bovine growth hormone (bGH) pre-mRNA undergoes alternative splicing in which the last intron is retained and transported to the cytoplasm. Our goal was to characterize the cis-acting signals in bGH pre-mRNA that collectively determine the distribution between intron splicing and intron retention. We now demonstrate that the balance between splicing and intron retention in cytoplasmic mRNA is primarily determined by the interaction of three splicing signals and the degree to which these signals deviate from consensus splicing signals. Intron retention requires the presence of both suboptimal 5'- and 3'-splice sites. Mutation of either splice site toward consensus leads to complete splicing of the intron. In the presence of both wild-type, suboptimal splice sites, efficient splicing of this intron is ensured by the presence of a third splicing element, a purine-rich exonic splicing enhancer (ESE). Although strong ESEs can be contained within very small sequences, the bGH ESE activity appears to be composed of multiple sequences spread throughout a 115-nucleotide region of exon 5. Consequently, the final ratio of splicing to intron retention depends on the balance between the relative strengths of each of these three splicing signals, which still allow intron-containing coding sequences to be transported to the cytoplasm.

Alternative Splicing↗

PubMed1995

Protein-induced folding of a group I intron in cytochrome b pre-mRNA.

Some group I introns have been shown to be self-splicing in vitro, but perhaps all require proteins for splicing in vivo. Sequence differences affect the stability of secondary structures and may explain why some group I introns function efficiently without protein cofactors while others require them. The terminal intron of the cytochrome b pre-mRNA from yeast mitochondria needs a nucleus-encoded protein for splicing, even though it splices autocatalytically in high salt in vitro. This system has the advantage that the protein is specific for this intron, and yet the structure of the catalytically active RNA can be studied in its absence. We have modified the intron by chemical and enzymatic treatment in the presence and absence of the protein to determine the impact of the protein on the secondary and tertiary structures of the intron. We found protein-induced formation of secondary and tertiary structures within the intron, and the same structures also form in high salt autocatalytic conditions. We have also studied UV cross-links to determine those bases of the intron that interact directly with the protein and found that the protein contacts the intron most intimately at the structures denoted P1, L2, P4, and P6a.

Base Sequence↗

PubMed2003

Discovery of a group I intron in the SSU rDNA of Ploeotia costata (Euglenozoa).

The gene coding for the small ribosomal subunit RNA of Ploeotia costata contains an actively splicing group I intron (Pco.S516) which is unique among euglenozoans. Secondary structure predictions indicate that paired segments P1-P10 as well as several conserved elements typical of group I introns and of subclass IC1 in particular are present. Phylogenetic analyses of SSU rDNA sequences demonstrate a well-supported placement of Ploeotia costata within the Euglenozoa; whereas, analyses of intron data sets uncover a close phylogenetic relation of Pco.S516 to S-516 introns from Acanthamoeba, Aureoumbra lagunensis (Stramenopila) and red algae of the order Bangiales. Discrepancies between SSU rDNA and intron phylogenies suggest horizontal spread of the group I intron. Monophyly of IC1 516 introns from Ploeotia costata, A. lagunensis and rhodophytes is supported by a unique secondary structure element: helix P5b possesses an insertion of 19 nt length with a highly conserved tetraloop which is supposed to take part in tertiary interactions. Neither functional nor degenerated ORFs coding for homing endonucleases can be identified in Pco.S516. Nevertheless, degenerated ORFs with His-Cys box motifs in closely related intron sequences indicate that homing may have occurred during evolution of the investigated intron group.

Acanthamoeba↗

PubMed2003

Variability and phylogenetic incongruence of an SSU nrDNA group I intron in Artomyces, Auriscalpium, and Lentinellus (Auriscalpiaceae: Homobasidiomycetes).

Previous research has shown that a group I intron occurs in the SSU ribosomal DNA gene of isolates of Artomyces (Clavicorona, in part) and Lentinellus, but apparently it is absent in an Auriscalpium isolate. However, further investigation revealed that the intron is apparently absent in some species of Artomyces and Lentinellus and is present in at least one species of Auriscalpium. To examine this further, the presence or absence of the group I intron is reported for 13 species of Lentinellus, two species of Auriscalpium, and 16 species of Artomyces. The presence of the intron among the species was variable and is documented for seven species of Lentinellus, one species of Auriscalpium, and 12 species of Artomyces. Furthermore, the presence of the intron was variable among the isolates of several species, and variability of its presence was observed within single isolates, indicating inter-ribosomal repeat heterogeneity. Independent phylogenetic estimations were generated for the intron and nuclear ribosomal internal transcribed spacer regions (ITS). Tests of congruence for the two trees indicated that the data were heterogeneous. Some of the discontinuity between the two phylogenies is due to placement of the Ar. austropiperatus intron within the Lentinellus intron clade. Variability in the length of the intron was observed in populations of the pan-Northern Temperate species Ar. pyxidatus. This was due to the presence of an additional unknown insertional element found only within North American collections of Ar. pyxidatus and absent from European and Asian collections.

Basidiomycota↗

PubMed1991

A mixed group II/group III twintron in the Euglena gracilis chloroplast ribosomal protein S3 gene: evidence for intron insertion during gene evolution.

The splicing of a 409 nucleotide intron from the Euglena gracilis chloroplast ribosomal protein S3 gene (rps3) was examined by cDNA cloning and sequencing, and northern hybridization. Based on the characterization of a partially spliced pre-mRNA, the intron was characterized as a 'mixed' twintron, composed of a 311 nucleotide group II intron internal to a 98 nucleotide group III intron. Twintron excision is via a 2-step sequential splicing pathway, with removal of the internal group II intron preceding excision of the external group III intron. Based on secondary structural analysis of the twintron, we propose that group III introns may represent highly degenerate versions of group II introns. The existence of twintrons is interpreted as evidence that group II introns were inserted during the evolution of Euglena chloroplast genes from a common ancestor with eubacteria, archaebacteria, cyanobacteria, and other chloroplasts.

Amino Acid Sequence↗

PubMed1992

Analysis of nonuniformity in intron phase distribution.

The distribution of different intron groups with respect to phases has been analyzed. It has been established that group II introns and nuclear introns have a minimum frequency of phase 2 introns. Since the phase of introns is an extremely conservative measure the observed minimum reflects evolutionary processes. A sample of all known, group I introns was too small to provide a valid characteristic of their phase distribution. The findings observed for the unequal distribution of phases cannot be explained solely on the basis of the mobile properties of introns. One of the most likely explanations for this nonuniformity in the intron phase distribution is the process of exon shuffling. It is proposed that group II introns originated at the early stages of evolution and were involved in the process of exon shuffling.

Animals↗

PubMed1992

A maturase-encoding group IIA intron of yeast mitochondria self-splices in vitro.

Intron 1 of the coxI gene of yeast mitochondrial DNA (aI1) is a group IIA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, aI1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4Cl or KCl or 2M (NH4)2SO4) for ready detection of splicing activity. In KCl-stimulated reactions, splicing occurs with little normal branch formation; a post-splicing reaction of linear excised intron RNA that forms shorter lariat RNAs with branches at cryptic sites was evident in those samples. At low levels of added NH4Cl or KCl, the precursor RNA carries out the first reaction step but appears blocked in the splicing step. AI1 RNA is most reactive at 37-42 degrees C, as compared with 45 degrees C for the group IIB introns; and it lacks the KCl- or NH4Cl-dependent spliced-exon reopening reaction that is evident for the self-splicing group IIB introns of yeast mitochondria. Like the group IIB intron aI5 gamma, the domain 4 of aI1 can be largely deleted in cis, without blocking splicing; also, trans-splicing of half molecules interrupted in domain 4 occurs. This is the first report of a maturase-encoding intron of either group I or group II that self-splices in vitro.

Base Sequence↗

PubMed1993

A complex twintron is excised as four individual introns.

Twintrons are introns-within-introns excised by sequential splicing reactions. A new type of complex twintron comprised of four individual group III introns has been characterized. The external intron is interrupted by an internal intron containing two additional introns. This 434 nt complex twintron within a Euglena gracilis chloroplast ribosomal protein gene is excised by four sequential splicing reactions. Two of the splicing reactions utilize multiple 5'- and/or 3'-splice sites. These findings are evidence that introns with multiple active splice sites can be formed by the repeated insertion of introns into existing introns.

Amino Acid Sequence↗

PubMed1997

RNA editing status of nad7 intron domains in wheat mitochondria.

The most highly conserved structures of group II introns are the helical domains V and VI near the 3'splice site. Within this region of each of the four introns in the wheat mitochondrial nad7 gene encoding NADH dehydrogenase subunit 7, there are A-C mispairs. To determine whether C-to-U type RNA editing restores conventional A-U pairing, we sequenced RT-PCR products from partially-spliced nad7 template RNA and gel-fractionated, excised intron RNA. We examined transcripts from germinating wheat embryos and seedlings because these two stages of development show pronounced differences in steady state levels of nad7 intronic RNAs. We observed editing at only two of the six predicted sites, and they were located at homologous positions within domain V of the third and fourth introns. A third site was found to be edited within the unmodelled domain VI loop of the fourth intron. Similar patterns of RNA editing were seen in wheat embryos and seedlings. These observations, and the presence of other non-conventional base pairs particularly within domain V of plant mitochondrial introns, indicate weaker helical core structure than in ribozymic group II introns. Moreover, the incompleteness or absence of editing in wheat nad7 excised intron RNA suggests that, although editing may contribute to splicing efficiency, it is not essential for splicing.

Base Composition↗

PubMed1997

Intronic and exonic sequences modulate 5' splice site selection in plant nuclei.

Pre-mRNA transcripts in a variety of organisms, including plants, Drosophila and Caenorhabditis elegans, contain introns which are significantly richer in adenosine and uridine residues than their flanking exons. Previous analyses using exonic and intronic replacements between two nonequivalent 5'splice sites in the 469 nt long rbcS3A intron 1 provided the first evidence indicating that, in both tobacco and Drosophila nuclei, 5'splice site selection is strongly influenced by the position of that site relative to the AU transition point between exon and intron. To differentiate between two potential models for 5'splice site recognition, we have expressed a completely different set of intronic and exonic replacement constructs containing identical 5'splice sites upstream of beta-conglycinin intron 4 (115 nt). Mutagenesis and deletion of the upstream 5'splice site demonstrate that intronic AU-rich sequences function by promoting recognition of the most upstream 5'splice site rather than by masking the downstream 5'splice site. Sequence insertions define a role for AG-rich exonic sequences in plant pre-mRNA splicing by demonstrating that an AG-rich element is capable of promoting downstream 5'splice site recognition. We conclude that AU-rich intronic sequences, AG-rich exonic sequences and the 5'splice site itself collectively define 5'intron boundaries in dicot nuclei.

Base Sequence↗

PubMed2003

Database for mobile group II introns.

Group II introns are self-splicing RNAs and retroelements found in bacteria and lower eukaryotic organelles. During the past several years, they have been uncovered in surprising numbers in bacteria due to the genome sequencing projects; however, most of the newly sequenced introns are not correctly identified. We have initiated an ongoing web site database for mobile group II introns in order to provide correct information on the introns, particularly in bacteria. Information in the web site includes: (1) introductory information on group II introns; (2) detailed information on subfamilies of intron RNA structures and intron-encoded proteins; (3) a listing of identified introns with correct boundaries, RNA secondary structures and other detailed information; and (4) phylogenetic and evolutionary information. The comparative data should facilitate study of the function, spread and evolution of group II introns. The database can be accessed at http://www.fp.ucalgary.ca/group2introns/.

Databases, Nucleic Acid↗

PubMed2005

Localization, mobility and fidelity of retrotransposed Group II introns in rRNA genes.

We previously showed that the group II Lactococcus lactis Ll.LtrB intron could retrotranspose into ectopic locations on the genome of its native host. Two integration events, which had been mapped to unique sequences, were localized in the present study to separate copies of the six L.lactis 23S rRNA genes, within operon B or D. Although further movement within the bacterial chromosome was undetectable, the retrotransposed introns were able to re-integrate into their original homing site provided on a plasmid. This finding indicates not only that retrotransposed group II introns retain mobility properties, but also that movement occurs back into sequence that is heterologous to the sequence of the chromosomal location. Sequence analysis of the retrotransposed introns and the secondary mobility events back to the homing site showed that the introns retain sequence integrity. These results are illuminating, since the reverse transcriptase (RT) of the intron-encoded protein, LtrA, has no known proofreading function, yet the mobility events have a low error rate. Enzymatic digests were used to monitor sequence changes from the wild-type intron. The results indicate that retromobility events have approximately 10(-5) misincorporations per nucleotide inserted. In contrast to the high RT error rates for retroviruses that must escape host defenses, the infrequent mutations of group II introns would ensure intron spread through retention of sequences essential for mobility.

Base Sequence↗

PubMed2006

Self-splicing of a group IIC intron: 5' exon recognition and alternative 5' splicing events implicate the stem-loop motif of a transcriptional terminator.

Bacterial IIC introns are a newly recognized subclass of group II introns whose ribozyme properties have not been characterized in detail. IIC introns are typically located downstream of transcriptional terminator motifs (inverted repeat followed by T's) or other inverted repeats in bacterial genomes. Here we have characterized the self-splicing activity of a IIC intron, B.h.I1, from Bacillus halodurans. B.h.I1 self-splices in vitro through hydrolysis to produce linear intron, but interestingly, additional unexpected products were formed that were highly dependent on ionic conditions. These products were determined to represent alternative splicing events at the 5' junction and cleavages throughout the RNA transcript. The alternative splicing and cleavage events occurred at cryptic splice sites containing stem-loop and IBS1 motifs, suggesting that the 5' exon is recognized by both elements. These results provide the first example of a group II intron that uses 5' splice sites nonadjacent to the ribozyme structure. Furthermore, the data suggest that IIC introns differ from IIA and IIB introns with respect to 5' exon definition, and that the terminator stem-loop substitutes in part for the missing IBS2-EBS2 (intron and exon binding sites 2) interaction.

Alternative Splicing↗

PubMed2002

Do introns favor or avoid regions of amino acid conservation?

Are intron positions correlated with regions of high amino acid conservation? For a set of ancient conserved proteins, with intronless prokaryotic but intron-containing eukaryotic homologs, multiple sequence alignments identified residues invariant throughout evolution. Intron positions between codons show no preferences. However, introns lying after the first base of a codon prefer conserved regions, markedly in glycines. Because glycines are in excess in conserved regions, this behavior could reflect phase-one introns entering glycine residues randomly in the ancestral sequences. Examination of intron positions within codons of evolutionarily invariable amino acids showed that roughly 50% of these introns are bordered by guanines at both 5'- and 3'-ends, 25% have a G only before the intron, and 5% have a G only after the intron, whereas about 20% are bordered by nonguanine bases.

Alternative Splicing↗

PubMed1992

Group I introns within the nuclear-encoded small-subunit rRNA gene of three green algae.

Four group I introns from the nuclear-encoded (18S) rRNA genes of three chlorophycean green algae are described; two are in Dunaliella parva, and one each is in D. salina and Characium saccatum. The introns within the gene in the latter two organisms are located at the sites equivalent to the 5' and 3' introns of D. parva, respectively. All four introns lack open reading frames and are relatively small, 381-447 bp. Both primary- and secondary-structural features place these introns within subgroup IC1 described by Michel and Westhof. Phylogenetic relationships of the three intron-containing taxa and their relatives, as inferred from comparisons of 18S rDNA sequences, suggest that inheritance of the introns along with the gene can account for their present distribution. The discovery of these four introns, in addition to two others known to exist in other chlorophycean green algae, suggests that group I introns within the 18S rRNA gene may be relatively common in the green algae.

Base Sequence↗