Evolution of small putative group I introns in the SSU rRNA gene locus of Phialophora species
© Rogers et al; licensee BioMed Central Ltd. 2011
Received: 29 January 2011
Accepted: 22 July 2011
Published: 22 July 2011
Group I introns (specifically subgroup IC1) are common in the nuclear ribosomal RNA genes of fungi. While most range in length from more than 200 to nearly 1800 nucleotides (nt) in length, several small putative (or degenerate) group I introns have been described that are between 56 and 81 nt. Although small, previously we demonstrated that the Pa SSU intron in the rRNA small subunit gene of Phialophora americana isolate Wang 1046 is capable of in vitro splicing using a standard group I intron pathway, thus qualifying it as a functional ribozyme.
Here, we describe eight short putative group I introns, ranging in length from 63 to 75 nt, in the rRNA small subunit genes of Phialophora isolates, a fungal genus that ranges from saprobic to pathogenic on plants and animals. All contain putative pairing regions P1, P7, and P10, as well as a pairing region formed between the middle of the intron and part of the 3' exon. The other pairing regions common in the core of standard group I introns are absent. However, parts of the 3' exon may aid in the stabilization of these small introns. Although the eight putative group I introns were from at least three species of Phialophora, phylogenetic analysis indicated that the eight are monophyletic. They are also monophyletic with the small introns of two lichen-forming fungi, Porpidia crustulata and Arthonia lapidicola.
The small putative group I introns in Phialophora have common features that may represent group I introns at their minima. They appear to have a single origin as indicated by their monophyly in phylogenetic analyses.
Group I introns are capable of self-splicing in vitro, and members of the IC1 subgroup are relatively common in the nuclear ribosomal RNA genes of fungi [1–3]. A few hypotheses have been proposed to explain how group I introns became established in the rRNA gene locus. Intron homing is one of the most likely mechanisms for converting an intron-less allele into one containing an intron, and has been demonstrated in experimental studies. This is initiated by the product of an intron-encoded homing endonuclease gene (HEG), which cleaves an intron-less allele at or near the intron insertion site [3–5]. This generates a double-stranded DNA break at the site of intron insertion. The intron-containing allele is used as the template to repair the break resulting in insertion of the DNA version of the intron and co-conversion of flanking exon sequences. However, very few group I introns contain open reading frames, indicating that the HEG's have been lost, they are elsewhere in the genome (and act in trans) or they are no longer functional in the genome rendering the introns immobile. Establishment of group I introns in the rRNA gene locus appears to have occurred tens to hundreds of millions of years ago, since the introns are found in phylogentically diverse organisms and the sequence diversity is large.
There are no known advantageous effects of group I introns to host organisms . They have been reported as selfish or parasitic genes that are adapted to assure their survival. However, some studies suggest that their widespread distribution indicates their evolutionary success and importance [7–10]. The presence of these introns in a broad range of species is due to a dynamic equilibrium between gains, mutations, and losses tempered by maintenance of accurate splicing of the exons. Both vertical and horizontal transmissions have been demonstrated, and transposition has been proposed as the mechanism that is responsible for movement of the introns within genomes [3–5, 9, 11].
In previous studies we reported the presence of self-splicing group I introns in the rRNA small subunit (SSU) genes of Cenococcum geophilum, as well as the location and self-splicing ability of a small (67 nucleotides, nt) group I intron in Phialophora americana Wang 1046 [13, 14]. This small intron may have originated from an unequal crossover in an ancestor that was amplified in the genome. Gene conversion, which is active in the rRNA gene locus, may have increased copy number of the mutant. Strong selective pressure is in effect in the rRNA locus to retain the production of the large number of rRNAs required in each cell. Therefore, the small introns must have retained their ability to splice in order to allow the production of functional small subunit rRNA. In this study we compared eight small putative group I introns from different isolates of Phialophora species (anamorphic dematiaceous fungi) in order to study the structure and evolution of these introns after their establishment in the rRNA genes. We show that the small introns differ in sequence, but retain similar structural features.
List of isolates and sequences used in this study.
Intron Length (nt)
Source of Isolate
Tilia sp., Virginia, USA, Conant 743
Paper pulp, Wisconsin, USA, Conant 333
Human foot lesion biopsy, Wisconsin, USA
Fraxinus sp., New York, USA, DAOM 64689
Decaying wood, New York, USA
Decaying wood, New York, USA
Human, Texas, USA
Multiple sequence analyses were performed to corroborate the presence and position of the introns in the isolates. Intron sequences were used to compare among the isolates. Sequences were aligned using CLUSTALW2 http://www.ebi.ac.uk/Tools/clustalw2/index.html. The alignments were examined, and manual adjustments were performed. Phylogenetic analyses were performed with PAUP (Phylogenetic Analysis Using Parsimony; ) using Maximum Parsimony, with bootstrapping.
The intron sequences were subjected to secondary structure analysis using Mfold (vers. 2.2, ). Because of the short lengths of the introns, after the initial determinations of secondary structure, parts of the sequences were removed and reanalyzed separately. Final manual adjustments were made in each case.
The Pa SSU intron (67 nt in isolate Wang 1046), located in the rRNA SSU gene of P. americana (Figure 1), is the smallest known intron with demonstrated group I ribozyme activity . Its size affords an opportunity to study the evolution of intron function. The mechanism of splicing differs slightly from that of standard group I introns. It proceeds through two trans-esterification reactions, as in the group I intron of Tetrahymena thermophila[23, 24], but certain variations have been found in the second reaction, in that it employs a U (an omega ωU) as the last nucleotide of the intron instead of the canonical ωG found in almost all other group I introns investigated to date . The other small introns described here are in the same location (nucleotide 1516, relative to E. coli) as the Pa SSU group I intron in isolate Wang 1046 (except the intron from Porpidia crustulata, which is at the 516 position ). While they all have similar secondary structure, their sequences differ.
Secondary structure conservation is evident in these small introns. Pairing regions P1, P7, and P10, as well as another pairing region between the middle of the introns and the 3' exons (termed PM) all were maintained in each of the introns (Figure 2). Furthermore, from our previous report , the P7 regions of these small introns probably originated as part of the P9.2 regions of larger group I introns. While the important secondary structures of these small introns are maintained, they alone may not be sufficient to hold the intron in the active tertiary structure for splicing to occur. Other parts of the rRNA gene locus may aid in this process. For example, in our previous in vitro splicing experiments, when RNA was used that included only the intron plus approximately 20 nt of each of the 5' and 3' exons, splicing was inefficient or failed. However, when a longer section (approximately 300 nt, including the entire ITS1) of the exon was included, splicing occurred in vitro. Therefore, part of the SSU and ITS1 rRNA may form the tertiary structure that maintains splicing activity in these small putative group I introns.
Ribosomal RNA genes are arranged in tandem arrays of 60-200 copies in fungi. Recombination and gene conversion act to homogenize the individual repeats. However, mutations can lead to a heterogeneous combination of sequences. In one case, an intron-less allele coexisted with intron-containing versions. Sequence results demonstrated that there often were one or more mutant versions of the introns in each of the isolates (not shown). While it is unknown whether the mutant introns are functional, some differed from the functional Pa SSU intron in isolate Wang 1046, and therefore they may be pseudogenes. Vertical transmissions, mutations and losses are common in the evolution of group I introns in fungi [10, 25]. The small putative introns in Phialophora, all appear to have originated from a single unequal crossover event that occurred in a common ancestor . This is supported by three characteristics: 1. All are found at a single nucleotide position in the rRNA SSU gene, 2. All have similar secondary structures, and 3. They form a single monophyletic clade.
Many studies have shown evidence for stable maintenance of group I introns over long periods of time [1, 26, 27], which seems to be the case with the small putative introns described here . Previous studies suggested the presence of group I introns sporadically in distant lineages of organisms that did not reflect the overall phylogeny. Those results suggest horizontal transmission between distinct lineages [3, 11, 28–30]. However, most of these involved large introns, the largest of which sometimes contained genes for homing endonucleases. No open reading frames that encode proteins for mobility such as homing endonucleases or reverse transcriptases have been reported in any Phialophora species. Certainly, a protein encoded from these small putative introns is implausible, and in general, open reading frames are absent from introns in the 1506-1521 location of the rRNA SSU genes in fungi. However, the different location for the Porpidia crustulata small degenerate intron (at position 516) suggests that it might have inserted into the rRNA locus through horizontal transfer or transposition.
Comparison of the Pa SSU (Wang 1046) intron with other introns inserted at the same site indicate that they are orthologs, although their sequences and lengths vary [1, 12, 13]. Group I introns inserted at different sites have been shown to be more distantly related even in the same species [3, 10, 31] and thus are likely to be paralogs. However, introns that are within the 1506-1521 region group together in phylogenetic analyses , and may represent a single insertion event, followed by mutation and use of cryptic splice sites. This creates the appearance of intron movement. However, the change in the splice site might not have been caused by translocation or transposition of the intron. The similarities in intron sequences among the isolates and the similarity to the other group I introns in the same region of the SSU rRNA gene, and the fact that they appear to be monophyletic  indicates that these introns moved into this location at a time that predated the separation of these taxa, which probably occurred at least tens of millions of years ago. Recombination, mutation, gene conversion, and vertical inheritance have resulted in the present distribution and diversity of group IC1 introns in the 1506-1521 region of the rRNA SSU genes in fungi, including the presence of these small putative group I introns.
The set of small putative group I introns from the SSU rRNA genes of Phialophora species are monophyletic, presumably having originated from a single mutational event. All are similar to the functional putative group I intron from P. americana isolate Wang 1046, in that each possess a P1, P7 and P10. Although their primary sequences have diverged, they all have a G·U pair at the 5' splice site, a G-C pair in the P7 guanosine site, an unpaired A on the 5' side of P7, and a U as the final nucleotide of the intron. These introns may represent the minimum size for group I ribozymes.
We thank Professor CJK Wang for providing the cultures for this study. This paper is dedicated to Professor Stephen Shuen-Shan Wang.
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