Terminal-Sequence Conservation Identifies Spliceosomal Introns in Ascomycete 18S RNA Genes

Oscar F. Cubero1,*, Paul D. Bridge{ddagger} and Ana Crespo*

*Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain; and
{dagger}School of Biological and Chemical Sciences, Birkbeck, University of London, London, England, and Mycology Section, Royal Botanic Garden, Kew, Richmond, England

Abstract

Twenty-four new insertions were obtained from seven different locations in the nuclear 18S rDNA for seven species of the lichen-forming fungal genus Physconia. They were analyzed allowing for terminal sequence conservation by adopting a flexible approach to exact insertion site position, and they were compared with 12 previously reported small insertion sequences from the 18S ribosomal RNA gene. Such insertions have previously been proposed to be degenerate self-splicing group I introns; however, the methodology used here identified consensus terminal sequences characteristic of spliceosomal introns. This finding is the first suggestion that multiple spliceosomal introns occur in ribosomal genes.

Introduction

Insertions in the nuclear small-subunit (SSU) rRNA gene have been described for many eukaryotic organisms. Such insertions occur in several discrete conserved positions (Gargas, DePriest, and Taylor 1995Citation ) and can be optional even at the population level (DePriest 1993Citation ; Crespo et al. 1997Citation ). Most SSU rRNA introns so far reported are typically longer than 180 bp and have been identified as group I introns on the basis of their conserved structures (Cech 1988Citation ; Gargas, DePriest, and Taylor 1995Citation ). The existence of introns and their distribution among organisms and genes have given rise to a number of theories of intron origin and evolution. These theories can be grouped around two main theoretical branches: the exon theory of genes, or early-intron theory (Blake 1978Citation ; Doolittle 1978Citation ), and the insertional theory of introns, or late-intron theory (Rogers 1989Citation ; Cavalier-Smith 1991Citation ).

In recent years, there have been reports of small a number of insertions of less than 100 bp at several points in the nuclear SSU rDNA of different groups of filamentous ascomycete fungi (Rogers et al. 1993Citation ; Gargas, DePriest, and Taylor 1995Citation ; Grube, Gargas, and DePriest 1996Citation ; Stenroos and DePriest 1998Citation ; Winka, Alberg, and Eriksson 1998Citation ). These short introns lack the necessary sequence and conserved structure to be considered group I introns, but in some cases sequences interpreted as internal guide sequences (IGSs) for splicing, similar to those in group I introns, have been described. These small insertions have been described at sites where group I introns have been found in other organisms (Gargas, DePriest, and Taylor 1995Citation ). Site conservation, together with the IGSs, has been used to interpret the short insertions from the ascomycetes Arthonia lapidicola, Phialophora americana, and Porpidia crustulata as degenerate group I introns (Gargas, DePriest, and Taylor 1995Citation ; Grube, Gargas, and DePriest 1996Citation ). The small intron sequences from these fungi can be folded to give a secondary structure similar to that of the P1 and P2 stems of group I introns. Similar small insertions from Cladonia, Stereocaulon, Cladina, and Physcia are also considered to contain the two-stem structure, but without an IGS sequence (Stenroos and DePriest 1998Citation ). One problem in accurately determining the origin of such small insertions is the short length of the sequences involved, along with the small number of such observations available.

The sequences of 24 new small insertions from the nuclear SSU rDNA of specimens of the lichen-forming ascomycete fungi Physconia were obtained. These were compared with the 12 previously reported similar short insertions (Rogers et al. 1993Citation ; Gargas, DePriest, and Taylor 1995Citation ; Grube, Gargas, and DePriest 1996Citation ; Stenroos and DePriest 1998Citation ; Winka, Ahlberg, and Eriksson 1998Citation ). The aim of the program was to identify any conserved sequences or structures that could provide indications as to the origin and significance of small insertions and the surrounding rRNA gene block.

Materials and Methods

A cetyl-trimethyl ammonium bromide (CTAB)–based extraction method (Cubero et al. 1999Citation ) was used to extract the total DNA from samples of Physconia detersa, Physconia distorta, Physconia elegantula, Physconia enteroxantha, Physconia grisea, Physconia perisidiosa, Physconia subpulverulenta, and Physconia venusta (collection details are indicated in table 1 ). Small portions of sample were ground in liquid nitrogen and incubated in CTAB extraction buffer (1% CTAB, 1 M NaCl, 100 mM Tris-HCl [pH 8.0], 20 mM EDTA, 1% polyvinyl polypyrrolidone) at 65°C. After two treatments with chloroform : iso-amylalcohol (CI, 24:1), DNA was precipitated by addition of two volumes of CTAB precipitation buffer (1% CTAB, 50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 40 mM NaCl) and resuspended in 1.2 M NaCl. After a second treatment with CI, DNA was precipitated by addition of 0.6 volumes of isopropanol and resuspended in water.


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Table 1 Collection Numbers, Small Insertion Sites, and Insertion Sequence Accession Numbers for the Physconia Samples Used in this Work

 
The major part of the SSU rDNA was amplified in two overlapping fragments with the fungal-specific primer pairs nu-SSU-0072-5'/nu-SSU-0852-3' and nu-SSU-0819-5'/nu-SSU-1750-3' (Gargas and DePriest 1996Citation ). The amplification conditions were a "hot start" at 94°C for 5 min followed by 35 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 2 min. The reaction was completed with a final 10-min extension step at 72°C. PCR products obtained from the nuclear SSU rDNA were sequenced, and the total sequences varied in length from approximately 1,800 to 3,200 bp. The product from P. subpulverulenta was 1,800 bp and did not contain any insertions. This sequence was therefore used as an initial guide to manually align and identify putative insertion elements.

PCR products were purified through commercial purification columns (Biotools) and sequenced using the PRISM Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit and a 373 Applied Biosystems automated sequencer. Primers used for sequencing reaction were those used in the PCR and the internal primers nu-SSU-0497-3' and nu-SSU-1465-3' (Gargas and DePriest 1996Citation ).

DNA sequences were assembled and manually aligned. Insertion sequences were detected by comparison with existing Physconia sequences known to lack introns and by comparison with the Saccharomyces cerevisiae nuclear SSU rDNA sequence (GenBank accession number J01353). Insertions were named according to the fungal sample and the insertion position with reference to the corresponding position in Escherichia coli (GenBank accession number J01695). Sequences obtained were submitted to the NCBI database under the accession numbers given in table 1 .

The specimens of P. distorta and P. perisidiosa were used for RNA isolation and amplification of the 18S rRNA to check for intron removal. Total RNA was isolated through RNA purification columns (Biotools). cDNA of the 18S rRNA was obtained by RT-PCR (Promega RT-PCR system). Product size was obtained by comparison with a 50-bp DNA ladder.

The hypothesis that insertions were degenerate group I introns was tested by obtaining base-pairing matrices from each alignment using the program DCSE (De Rijk and De Wachter 1993Citation ). This method searches for compensatory segments within the alignment and identifies compensatory mutations which can indicate putative conserved secondary structures.

The sequences of 12 previously reported short insertions from fungal nuclear SSU rDNA (Rogers et al. 1993Citation ; Gargas, DePriest, and Taylor 1995Citation ; Grube, Gargas, and DePriest 1996Citation ; Stenroos and DePriest 1998Citation ) were compared with those obtained in this study in an attempt to identify common conserved sequences and structures. The additional fungal species and GenBank accession numbers for the small insertions were as follows: Lecanora dispersa, L37734; Porpidia crustulata, L37735; Cladonia bellidiflora, U64682; Cladonia diversa, U64683; Cladina portentosa, U64684; Stereocaulon taeniarum, U64687; Stereocaulo tomentosum, U64688; Pertusaria trachythallina, U64685; Graphis scripta, AF038878; and Physcia aipolia, U64686. The Phialophora americana insertion sequence was obtained from Rogers et al. (1993)Citation , and the Arthonia lapidicola insertion sequence was obtained from Grube, Gargas, and DePriest (1996)Citation .

Results and Discussion

Total length of nuclear SSU rDNA varied from approximately 1,720 to 3,200 bp. The product from P. subpulverulenta was 1,720 bp and did not contain any insertions. This sequence was therefore used as an initial guide to align and identify putative insertion elements. Fifty insertions, located at 15 different positions, were found in the remaining samples. Twenty-six of these insertions were typical group I introns (data not shown). Twenty-four small insertions (50–70 bp) were too short to be identified as group I introns, and these were recovered from seven different positions in different species (table 1 ).

The elimination of the insertions from the mature 18S RNA was verified by reverse transcription and amplification of the 18S cDNA in P. distorta and P. perisidiosa. Six small insertions and five group I introns or three small insertions and six group I introns had previously been detected in these specimens. The RT-PCR products obtained in both cases were 1,720 bp. As this was the expected size of the amplified segment when introns were not present, it seems likely that both the group I introns and the small insertions were removed during the maturation process.

The exact site of small insertion could not always be determined solely by comparison with the insertion-lacking sequence of P. subpulverulenta. This uncertainty was due to the difficulty in identifying the 5' and 3' termini of the insertion within the sequence. This can be illustrated by considering a theoretical sequence containing an insertion CACGG(N)nAGCG and the corresponding intron-lacking sequence CACGCG. In this case, there are two different points at which the insertion could be located depending on the identity of the first and last base in the insertion sequence: CAC[GG(N)nA]GCG or CACG[G(N)nAG]CG. In order to accurately align the short insertions, they were all considered with both alternative termini. The main criterion used in this alignment was to conserve the insertion sequence wherever possible. Under this criterion, all short insertions were found to have GT at their 5' termini and AG at their 3' termini. The final intron positions are given in table 1 , and these correspond to the positions after nucleotides 296, 297, 330, 513, 673, 943, and 1129 in E. coli.

There is considerable sequence divergence between the different insertion sequences. Insertions from different locations in the 18S rDNA are not alignable, except for those at locations 296 and 297, which are similar in sequence (50% similar positions between all of the sequences and 60%–83% similarity between pairs of sequences) and alignable. These insertions are interpreted as the result of an insertion sliding due to compensatory mutations at both intron-exon junctions (fig. 1 ), which is in agreement with the phylogenetic relationship between the species of the genus based on internal transcribed spacer sequences (unpublished data). For that reason, insertions at position 296 and insertions at position 297 are considered homologous in this work and are analyzed together.



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Fig. 1.—As insertions at locations 296 and 297 are similar in sequence, a common origin is suggested. The different insertion sites could be derived by a sliding of the intron due to compensatory mutations. This idea is congruent with the relationships between the Physconia species, as P. detersa and P. enteroxantha are closely related species, as are P. elegantula and P. perisidiosa. The possible sliding of the intron from position 297 to position 296 is represented, although sliding from position 296 to position 297 is equally possible

 
Insertions at the same location are alignable and similar in sequence, with the percentage similarity between pairs of insertions at the same location ranging from 58% to 87%. Percentages of similarity between all of the insertions at a given location were 42%, 78%, 53%, 57%, and 52% for insertions at locations 330, 513 (only two insertions found), 673, 943, and 1229, respectively. Sequence alignments are represented in figure 2 in the form of logos (Schneider and Stephens 1990Citation ), where the proportion of nucleotides at a given position is shown graphically.



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Fig. 2.—Logos showing the information at each position obtained from the alignments of the insertions at the six locations reported in this work (top to bottom: 297, 329, 513, 673, 943, and 1129), generated using the program Weblogos at http://www.bio.cam.ac.uk/seqlogo. The first and last three positions correspond with exon-bordering sequences. The height of each particular character is proportional to its frequency at a position. The relative height of each logo is proportional to the number of sequences used for its construction

 
In spite of the high divergence between insertions from different locations, there are a number of common features and conserved motifs that are present in all of them. These motifs are (1) GT at the 5' terminus, (2) AG at the 3' terminus, and (3) the sequence CTAAC, which is present about 9–13 bp upstream of the 3' end of the insertion. These three conserved motifs are all typical features of nuclear-encoded spliceosomal introns (Cech 1983Citation ; Raymond and Rosbash 1992Citation ; Stephens and Schneider 1992Citation ) and are involved in sequence recognition during the splicing process. The 5'- and 3'-terminal motifs are considered an almost universal characteristic of eukaryotic spliceosomal introns and are directly involved in the splicing process, in which they are recognized by the small nuclear ribonucleic particles U1 and U5 of the spliceosome (Lerner et al. 1980Citation ; Rogers and Wall 1980Citation ; Mount et al. 1983Citation ; Reich et al. 1992Citation ). The CTAAC motif is also involved in splicing and has been referred to as the "branch point" (Langford and Gallwitz 1983Citation ). This motif is recognized by the small nuclear ribonucleic particle U2 of the spliceosome (Wu and Manley 1989Citation ; Zhuang and Weiner 1989Citation ).

Some other features observed in the Physconia insertions are also characteristic of spliceosomal introns. These include the position of the putative branch point from the 3' end of the sequence, the high frequency of G at position 5 of the intron sequence (Cech 1983Citation ), and the presence of a pyrimidine immediately before the terminal AG motif (Jeffeys and Flavell 1977Citation ). Although these features are not all universal for spliceosomal introns, they occur at high frequencies in messenger RNA introns and are usually included in consensus descriptions.

A general consensus description for the small introns in Physconia nuclear SSU rDNA can be given as GT(A/T)N(G/A) ... PurCTAAC ... PyrAG, where G/A represents a purine that is usually G but occasionally A. The sequence and motif conservation described here provides evidence to support the hypothesis that the small insertions found in the SSU rDNA gene of the Physconia samples are putative spliceosomal introns. They possess conserved functional features required for external splicing in messenger preRNAs. This is the first suggestion that multiple insertions in the ribosomal genes are putative spliceosomal introns.

Ribosomal RNA genes generally lack introns, but in some organisms these genes contain large insertions which have been identified as group I introns (for review, see Gargas et al. 1995Citation ; Johansen, Muscarella, and Vogt 1996Citation ). Prior to this study, only 12 nuclear SSU rDNA small insertions had been described, all of them in ascomycete fungi (fig. 3 ). Of these, nine have been found in different species of the lichen-forming family lecanorales (Gargas, DePriest, and Taylor 1995Citation ; Stenroos and DePriest 1998Citation ), one has been found in the lichen-forming fungi A. lapidicola (arthoniales; Grube, Gargas, and DePriest 1996Citation ) and G. scripta (ostropales; Winka, Ahlberg, and Eriksson 1998Citation ), and one has been found in the parasitic fungus P. americana (dothideales; Rogers et al. 1993Citation ). The latter is the only small insertion in the nuclear SSU rDNA described as being a putative spliceosomal type. However, the possibility that further small SSU rRNA insertions may be spliceosomal introns has not been generally discussed. In practice, most workers have placed prime importance on potential secondary structures and insertion site, rather than on sequence conservation. As a result, there have been few, if any, comparisons of possible alternative insertion sites and little critical examination of sequence features.



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Fig. 3.—Putative conserved motifs in the sequences of 12 previously published insertions in the 18S SSU rDNA.

 
A careful examination of the previously published sequences of small introns in the 18S rDNA shows that there are multiple possible interpretations for the terminal sequences, at least one of which allows for typical spliceosomal GT/PyrAG termini (fig. 2 ). Additionally, most of these insertions have internal sequences that coincide with or resemble the PurCTAAG branch point sequence and have G as the fifth nucleotide. The P. crustulata small insertion differs in that the 3' terminus is GG and the internal conserved sequence is CTGAC. However, in both cases there would appear to be a transition from A to G. In practice, this would be of little significance, as the original matching U can form hydrogen bonds with G, and so the sites may still function. The small insertion from L. dispersa appears to have GAT/CT or AT/CTG termini (depending on which of the two different possible sites is considered) and GCTGAC as the putative internal sequence which also resembles the conserved described sequences in spliceosomal introns.

Putative Secondary Structures
In order to consider the possibility of conserved secondary structures among the Physconia small insertions, complementary segments were searched in the alignment. As the hypothesis of "degenerate group I introns" involves interactions between internal sequences of the intron- and exon-bordering sequences (Grube, Gargas, and DePriest 1996Citation ), 10 nucleotides before and after the insertion were included in the analysis.

The results obtained did not show any evidence for the existence of conserved or similar structures in the different Physconia small introns. Attempts to generate two-stem secondary structures or detect an IGS sequence as proposed for the small introns of A. lapidicola and P. crustulata (Grube, Gargas, and DePriest 1996Citation ) failed to give positive results, and although two-stem secondary structures were obtained by hand for some particular intron sequences, they were not in agreement with the conservation patterns shown in the alignments.

Distribution and Evolution of 18S rDNA Small Introns
Traditionally, ribosomal RNA genes are thought to lack spliceosomal introns of the type found in messenger RNAs. However, we have demonstrated that in some organisms there are introns in the 18S rDNA which clearly resemble those of protein spliceosomal introns. Currently, there is not enough information to establish a hypothesis about the origin of these ribosomal introns. However, any future hypothesis will need to consider several points:

  • The SSU small introns are eliminated from the mature rRNA and contain those motifs known to be functional in the splicing. It is therefore reasonable to assume that they may be related to or derived from spliceosomal introns and that some ribosomal genes have an intron-exon structures similar to those of protein genes.
  • The 18S small introns are rare. At present, although there are a large number of ribosomal sequences available from a broad range of organisms, small insertions have been observed in only a small number of organisms. All of these are ascomycete fungi and all of them, except for P. americana, are lichenized fungi. There is no taxonomic or evolutionary model available which would allow the small-insertion-containing fungi to be grouped separate from groups lacking introns. As a result, a hypothesis based on multiple intron acquisition in some organisms or intron loss in most organisms during evolution would be required to explain such a distribution.
  • Closely related species, such as those analyzed in the genus Physconia, contain multiple small introns which are in different combinations. This would tend to indicate that the loss or gain of introns has occurred recently in evolutionary time. However, there is no current phylogenetic model available for the species of Physconia analyzed for which the small intron distribution could be mapped and which could help in the inference of the direction of the process.
  • Many of the organisms in which small introns in the 18S rDNA have been described also contain group I introns. In Physconia (this work), Cladonia, Cladina, and Lecanora (Gargas, DePriest, and Taylor 1995Citation ; Stenroos and DePriest 1998Citation ), multiple group I introns have been found in the 18S rDNA. In P. americana, they have not been observed, but a group I intron has been described in the 18S rDNA of Phialophora gregata (Chen, Gray, and Grau 1998Citation ), and we have also observed group I introns in species of Physcia (unpublished results). Although the hypothesis of "degenerated group I introns" tries to establish a relationship between both intron types, we have not found any evidence to support that supposition for the Physconia small introns.
  • The results of this study show that maximizing terminal sequence conservation provides sufficient evidence to identify the small insertion from the SSU rRNA genes of filamentous fungi as putative spliceosomal introns. However, further knowledge of the wider distribution of these ribosomal small insertions is required before a full hypothesis can be developed.

    Acknowledgements

    We thank F. García-Olmedo, D. L. Hawksworth, J. Valcárcel, P. Vargas, and two anonymous reviewers for their useful comments. This work was supported by grants CAM-07B-0014-1997-97 from the Comunidad Autónoma de Madrid and APC-960082 from the Spanish Ministerio de Educación y Cultura (MEC). O.F.C. was supported by an FPI fellowship from the MEC.

    Footnotes

    Thomas Eickbush, Reviewing Editor

    2 Keywords: ribosomal insertion spliceosomal intron fungal insertion group I intron degenerate introns Physconia. Back

    1 Address for correspondence and reprints: Oscar F. Cubero, Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid 28040, Spain.E-mail: oscarhfc{at}retemail.es Back

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    Accepted for publication January 17, 1999.