A structural and phylogenetic analysis of the group IC1 introns in the order Bangiales (Rhodophyta)

Our previous study of the North American biogeography of Bangia revealed the presence of two introns inserted at positions 516 and 1506 in the nuclear-encoded SSU rRNA gene. We subsequently sequenced nuclear SSU rRNA in additional representatives of this genus and the sister genus Porphyra in order to examine the distribution, phylogeny, and structural characteristics of these group I introns. The lengths of these introns varied considerably, ranging from 467 to 997 nt for intron 516 and from 509 to 1,082 nt for intron 1506. The larger introns contained large insertions in the P2 domain of intron 516 and the P1 domain of intron 1506 that correspond to open reading frames (ORFs) with His-Cys box homing endonuclease motifs. These ORFs were found on the complementary strand of the 1506 intron in Porphyra fucicola and P. umbilicalis (HG), unlike the 516 intron in P. abbottae, P. kanakaensis, P. tenera (SK), Bangia fuscopurpurea (Helgoland), and B. fuscopurpurea (MA). Frameshifts were noted in the ORFs of the 516 introns in P. kanakaensis and B. fuscopurpurea (HL), and all ORFs terminated prematurely relative to the amino acid sequence for the homing endonuclease I-Ppo I. This raises the possibility that these sequences are pseudogenes. Phylogenies generated using sequences of both introns and the 18S rRNA gene were congruent, which indicated long-term immobility and vertical inheritance of the introns followed by subsequent loss in more derived lineages. The introns within the florideophyte species Hildenbrandia rubra (position 1506) were included to determine relationships with those in the Bangiales. The two sequences of intron 1506 analyzed in Hildenbrandia were positioned on a well-supported branch associated with members of the Bangiales, indicating possible common ancestry. Structural analysis of the intron sequences revealed a signature structural feature in the P5b domain of intron 516 that is unique to all Bangialean introns in this position and not seen in intron 1506 or other group IC1 introns.     

Multiple Group I Introns in the Small-Subunit rDNA of Botryosphaeria dothidea: Implication for Intraspecific Genetic Diversity

Botryosphaeria dothidea is a widespread and economically important pathogen on various fruit trees, and it often causes die-back and canker on limbs and fruit rot. In characterizing intraspecies genetic variation within this fungus, group I introns, rich in rDNA of fungi, may provide a productive region for exploration. In this research, we analysed complete small subunit (SSU) ribosomal DNA (rDNA) sequences of 37 B. dothidea strains, and found four insertions, designated Bdo.S943, Bdo.S1199-A, Bdo.S1199-B and Bdo.S1506, at three positions. Sequence analysis and structure prediction revealed that both Bdo.S943 and Bdo.S1506 belonged to subgroup IC1 of group I introns, whereas Bdo.S1199-A and Bdo.S1199-B corresponded to group IE introns. Moreover, Bdo.S1199-A was found to host an open reading frame (ORF) for encoding the homing endonuclease (HE), whereas Bdo.S1199-B, an evolutionary descendant of Bdo.S1199-A, included a degenerate HE. The above four introns were novel, and were the first group I introns observed and characterized in this species. Differential distribution of these introns revealed that all strains could be separated into four genotypes. Genotype III (no intron) and genotype IV (Bdo.S1199-B) were each found in only one strain, whereas genotype I (Bdo.S1199-A) and genotype II (Bdo.S943 and Bdo.S1506) occurred in 95% of the strains. There is a correlation between B. dothidea genotypes and hosts or geographic locations. Thus, these newly discovered group I introns can help to advance understanding of genetic differentiation within B. dothidea.     

EVIDENCE FOR LATERAL TRANSFER OF AN IE INTRON BETWEEN FUNGAL AND RED ALGAL SMALL SUBUNIT RRNA GENES1

A previous study of the North American biogeography of the red algal genus Hildenbrandia noted the presence of group I introns in the nuclear small subunit (SSU) rRNA gene of the marine species H. rubra (Sommerf.) Menegh. Group IC1 introns have been previously reported at positions 516 and 1506 in the nuclear SSU RNA genes in the Bangiales and Hildenbrandiales. However, the presence of an unclassified intron at position 989 in a collection of H. rubra from British Columbia was noted. This intron is a member of the IE subclass and is the first report of this intron type in the red algae. Phylogenetic analyses of the intron sequences revealed a close relationship between this IE intron inserted at position 989 and similar fungal IE introns in positions 989 and 1199. The 989 IE introns formed a moderately to well‐supported clade, whereas the 1199 IE introns are weakly supported. Unique structural helices in the P13 domain of the 989 and 1199 IE introns also point to a close relationship between these two clades and provide further evidence for the value of secondary structural characteristics in identifying homologous introns in evolutionarily divergent organisms. The absence of the 989 IE intron in all other red algal nuclear SSU rRNA genes suggests that it is unlikely that this intron was vertically inherited from the common ancestor of the red algal and fungal lineages but rather is the result of lateral transfer between fungal and red algal nuclear SSU rRNA genes.     

Terminal-sequence conservation identifies spliceosomal introns in ascomycete 18S RNA genes

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.     

The molecular evolution and structural organization of self-splicing group I introns at position 516 in nuclear SSU rDNA of myxomycetes

Group I introns are relatively common within nuclear ribosomal DNA of eukaryotic microorganisms, especially in myxomycetes. Introns at position S516 in the small subunit ribosomal RNA gene are particularly common, but have a sporadic occurrence in myxomycetes. Fuligo septica, Badhamia gracilis, and Physarum flavicomum, all members of the family Physaraceae, contain related group IC1 introns at this site. The F. septica intron was studied at the molecular level and found to self-splice as naked RNA and to generate full-length intron RNA circles during incubation. Group I introns at position S516 appear to have a particularly widespread distribution among protists and fungi. Secondary structural analysis of more than 140 S516 group I introns available in the database revealed five different types of organization, including IC1 introns with and without His-Cys homing endonuclease genes, complex twin-ribozyme introns, IE introns, and degenerate group I-like introns. Both intron structural and phylogenetic analyses indicate a multiple origin of the S516 introns during evolution. The myxomycete introns are related to S516 introns in the more distantly related brown algae and Acanthamoeba species. Possible mechanisms of intron transfer both at the RNA- and DNA-levels are discussed in order to explain the observed widespread, but scattered, phylogenetic distribution.     

Evolutionary dynamics of multiple group I introns in nuclear ribosomal RNA genes of endoparasitic fungi of the genus Cordyceps

A large number of group I introns were discovered in coding regions of small and large subunits of nuclear ribosomal RNA genes (SSU rDNA and LSU rDNA) in ascomycetous fungi of the genus CORDYCEPS: From 28 representatives of the genus, we identified in total 69 group I introns which were inserted at any of four specific sites in SSU rDNA and four specific sites in LSU rDNA. These group I introns reached sizes of up to 510 bp, occurred in up to eight sites in the same organism, and belonged to either subgroup IB3 or subgroup IC1 based on their sequence and structure. Introns inserted at the same site were closely related to each other among Cordyceps fungi, whereas introns inserted at different sites were phylogenetically distinct even in the same species. Mapped on the host phylogeny, the group I introns were generally not restricted to a particular lineage, but, rather, widely and sporadically distributed among distinct lineages. When the phylogenetic relationships of introns inserted at the same site were compared with the phylogeny of their hosts, the topologies were generally significantly congruent to each other. From these results, the evolutionary dynamics of multiple group I introns in Cordyceps fungi was inferred as follows: (1) most of the group I introns were already present at the eight sites in SSU and LSU rDNAs of the ancestor of the genus Cordyceps; (2) the introns have principally been immobile and vertically transmitted throughout speciation and diversification of Cordyceps fungi, which resulted in the phylogenetic congruence between the introns at the same site and their hosts; (3) in the course of vertical transmission, the introns have repeatedly been lost in a number of lineages independently, which has led to the present sporadic phylogenetic distribution of the introns; and (4) a few acquisitions of new introns, presumably through horizontal transmission, were identified in the evolutionary history of the genus Cordyceps, while no transpositions were detected. Losses of group I introns in SSU rDNA have occurred at least 27 times in the evolutionary course of the 28 Cordyceps members.     

Sequence Insertions and ITS Data Provide Congruent Information on Roccella canariensis and R. tuberculata (Arthoniales, Euascomycetes) Phylogeny

Four Roccella species, R. canariensis, R. fimbriata, R. montagnei, and R. tuberculata, were found to possess sequence insertions in up to four locations in the first half of the SSU rDNA. Insertions from one of these positions have been classified as group I introns, while the others may represent degenerative forms of group I introns or messenger RNA introns. Two of the insertion-containing taxa, R. canariensis and R. tuberculata, differ only in their dispersal strategy: R. canariensis is sexual, producing only fruiting bodies and R. tuberculata is sterile, producing only vegetative propagules, i.e., soredia. Because insertions occurred in specimens of both taxa, they were used to examine the phylogenetic relationships between and within the two species. The sequence insertions from each of the four positions were aligned and cladistically analyzed separately. Internal transcribed spacers (ITS) were additionally sequenced to study the phylogeny of all R. canariensis and R. tuberculata specimens. Three other Roccella species (R. babingtonii, R. fimbriata, and R. montagnei) and Dirina catalinariae were used as outgroups in this parsimony analysis. Sequence insertions were found to be potentially useful in phylogenetic studies, although due to the sequence dissimilarity, homology relations were difficult to establish above the species level and in some cases even within the species. The phylogenies obtained from the insertion matrices were totally consistent with the ITS data and the insertions were concluded to have been inherited. When the insertion and ITS data were combined for total evidence, R. canariensis and R. tuberculata did not form distinct lineages in the phylogenetic tree, but appeared mixed in well-supported groups containing both sorediate and fertile specimens.     

Patterns of Group I Intron Presence in Nuclear SSU rDNA of the Lichen Family Parmeliaceae

Group I introns are commonly reported within nuclear SSU ribosomal DNA of eukaryotic micro-organisms, especially in lichen-forming fungi. We have studied the primary and secondary structure of 70 new nuclear SSU rDNA group I introns of Parmeliaceae (Ascomycota: Lecanorales) and compared them with those available in databases, covering more than 60 species. The analyzed samples of Parmeliaceae fell into two groups, one having an intron at the 1506 site and another lacking this one but having another at the 1516 or 1521 position. Introns at the 1521 position seem to be transposed from 1516 sites. Introns at the 1516 position were similar in structure to ones previously reported at this site and known from other lecanoralean fungi, while those at the 1506 position showed structural differences and no similar introns are known from related fungi. The study of the distribution of group I introns within a large monophyletic ensemble of fungi has revealed an unexpected correlation between intron types and ecological and geographical parameters. The introns at the 1516 position occurred in mainly arctic, boreal, and temperate lichens, while those at position 1506 were present in mainly tropical and subtropical to oceanic mild-temperate taxa. Further, the 1516 introns occurred in genera with few distributed species that could represent older taxa, while the 1506 ones were mainly in species-rich genera that could be of recent speciation, as many species have wide distribution areas. The transition between two different environments has been accompanied by a change in introns gained and lost. [Reviewing Editor: Dr. Debashish Bhattacharya]     

The molecular evolution and structural organization of group I introns at position 1389 in nuclear small subunit rDNA of myxomycetes

The number of nuclear group I introns from myxomycetes is rapidly increasing in GenBank as more rDNA sequences from these organisms are being sequenced. They represent an interesting and complex group of intervening sequences because several introns are mobile (or inferred to be mobile) and many contain large and unusual insertions in peripheral loops. Here we describe related group I introns at position 1389 in the small subunit rDNA of representatives from the myxomycete family Didymiaceae. Phylogenetic analyses support a common origin and mainly vertical inheritance of the intron. All S1389 introns from the Didymiaceae belong to the IC1 subclass of nuclear group I introns. The central catalytic core region of about 100 nt appears divergent in sequence composition even though the introns reside in closely related species. Furthermore, unlike the majority of group I introns from myxomycetes the S1389 introns do not self-splice as naked RNA in vitro under standard conditions, consistent with a dependence on host factors for folding or activity. Finally, the myxomycete S1389 introns are exclusively found within the family Didymiaceae, which suggests that this group I intron was acquired after the split between the families Didymiaceae and Physaraceae.     

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.     

Nucleotide sequence of nuclear 5.4 S RNA of mouse cells

The nucleotide sequence of nuclear 5.4 S RNA, a new species of small nuclear RNA (snRNA) of mouse cells, was determined. The 5.4 S RNA consists of 138 nucleotide residues containing 1 mol each of 2,2,7- trimethylguanosine (m3(2,2,7) G), 2'-O-methyladenosine (Am), 2'-O-methyluridine (Um) and pseudouridine as modified nucleosides. This RNA has a cap structure, m3(2,2,7) ++GpppAm -, at its 5'-terminus and sequences complementary to the terminal consensus sequences of introns. The sequence complementary to the 5'-splice junction, A-U-C-C-psi-U-A-C-C-U-G, is very similar to the 5'-terminal sequence of U1 RNA.     

Naegleria nucleolar introns contain two group I ribozymes with different functions in RNA splicing and processing.

We have characterized the structural organization and catalytic properties of the large nucleolar group I introns (NaSSU1) of the different Naegleria species N. jamiesoni, N. andersoni, N. italica, and N. gruberi. NaSSU1 consists of three distinct RNA domains: an open reading frame encoding a homing-type endonuclease, and a small group I ribozyme (NaGIR1) inserted into the P6 loop of a second group I ribozyme (NaGIR2). The two ribozymes have different functions in RNA splicing and processing. NaGIR1 is an unusual self-cleaving group I ribozyme responsible for intron processing at two internal sites (IPS1 and IPS2), both close to the 5' end of the open reading frame. This processing is hypothesized to lead to formation of a messenger RNA for the endonuclease. Structurally, NaGIR2 is a typical group IC1 ribozyme, catalyzing intron excision and exon ligation reactions. NaGIR2 is responsible for circularization of the excised intron, a reaction that generates full-length RNA circles of wild-type intron. Although it is only distantly related in primary sequence, NaSSU1 RNA has a predicted organization and function very similar to that of the mobile group I intron DiSSU1 of Didymium, the only other group I intron known to encode two ribozymes. We propose that these twin-ribozyme introns define a distinct category of group I introns with a conserved structural organization and function.     

Association of a group I intron with its splice junction in 50S ribosomes: implications for intron toxicity.

The effect of genetic context on splicing of group I introns is not well understood at present. The influence of ribosomal RNA conformation on splicing of rDNA introns in vivo was investigated using a heterologous system in which the Tetrahymena group I intron is inserted into the homologous position of the Escherichia coli 23S rRNA. Mutations that block splicing in E. coli result in accumulation of unspliced 23S rRNA that is assembled into 50S complexes, but not 70S ribosomes. The data indicate that accommodation of the intron structure on the surface of the 50S subunit inhibits interactions with the small ribosomal subunit. Spliced intron RNA also remains noncovalently bound to 50S subunits on sucrose gradients. This interaction appears to be mediated by base pairing between the intron guide sequence and the 23S rRNA, because the fraction of bound intron RNA is reduced by point mutations in the IGS or deletion of the P1 helix. Association of the intron with 50S subunits correlates with slow cell growth. The results suggest that group I introns have the potential to inhibit protein synthesis in prokaryotes by direct interactions with ribosomes.     

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.     

Eu-Chlorella large subunit rDNA sequences and group I introns in ribosomal DNA of the paramecian symbiotic alga NC64A

Although the examination of large subunit ribosomal RNA genes (LSU rDNA) is advanced in phylogenetic studies, no corresponding sequence data from trebouxiophytes have been published, with the exception of ‘Chlorella’ellipsoidea Gerneck. We determined the LSU rDNA sequence of Chlorella vulgaris Beijerinck and of the symbiotic alga of green paramecium, Chlorella sp. NC64A. A total of 59 nucleotide substitutions were found in the LSU rDNA of the two species, which are disproportionately distributed. Primarily, 65% of the substitutions were encountered in the first 800 bp of the alignment. This segment apparently has evolved eight times faster than the complete SSU rDNA sequence, making it a good candidate for a phylogenetic marker and giving a resolution level intermediate between small subunit (SSU) rDNA and internal transcribed spacers. Green algae are known as a group I intron‐rich group along with rhodophytes and fungi. NC64A is particularly rich in the introns; five introns were newly identified from the LSU rDNA sequence, which we named Cnc.L200, Cnc.L1688, Cnc.L1926, Cnc.L2184 and Cnc.L2437, following the insertion positions. In the present study we analyzed these introns with three others (Cnc.S943, Cnc.S1367 and Cnc.S1512) that had already been found in NC64A SSU rDNA. Secondary structure modeling placed these introns in the group I intron family, with four introns belonging to subgroup C1 and the other four introns belonging to subgroup E. Five of the intron insertion positions are unique to the paramecian symbiont, which may indicate relatively recent events of intron infections that includes transpositions. Intron phylogeny showed unprecedented relationships; four Cnc. IC1 introns made a clade with some green algal introns with insertions at nine different positions, whereas four Cnc. IE introns made a clade with the S651 intron (Chlorella sp. AN 1–3), which lay as a sister to the S516 insertion position subfamily.     

Molecular evolution of pteridophytes and their relationship to seed plants: Evidence from complete 18S rRNA gene sequences

Complete 18S ribosomal RNA sequence data from representatives of all extant pteridophyte lineages together with RNA sequences from different seed plants were used to infer a molecular phylogeny of vascular plants that included all major land plant lineages. The molecular data indicate that lycopsids are monophyletic and are the earliest diverging group within the vascular land plants, whereasPsilotum nudum is more closely related to the seed plants than to other pteridophyte lineages. The phylogenetic trees based on maximum likelihood, parsimony and distance analyses show substantial agreement with the evolutionary relationships of land plants as interpreted from the fossil record.     

The evolution of homing endonuclease genes and group I introns in nuclear rDNA

Group I introns are autonomous genetic elements that can catalyze their own excision from pre-RNA. Understanding how group I introns move in nuclear ribosomal (r)DNA remains an important question in evolutionary biology. Two models are invoked to explain group I intron movement. The first is termed homing and results from the action of an intron-encoded homing endonuclease that recognizes and cleaves an intronless allele at or near the intron insertion site. Alternatively, introns can be inserted into RNA through reverse splicing. Here, we present the sequences of two large group I introns from fungal nuclear rDNA, which both encode putative full-length homing endonuclease genes (HEGs). Five remnant HEGs in different fungal species are also reported. This brings the total number of known nuclear HEGs from 15 to 22. We determined the phylogeny of all known nuclear HEGs and their associated introns. We found evidence for intron-independent HEG invasion into both homologous and heterologous introns in often distantly related lineages, as well as the "switching" of HEGs between different intron peripheral loops and between sense and antisense strands of intron DNA. These results suggest that nuclear HEGs are frequently mobilized. HEG invasion appears, however, to be limited to existing introns in the same or neighboring sites. To study the intron-HEG relationship in more detail, the S943 group I intron in fungal small-subunit rDNA was used as a model system. The S943 HEG is shown to be widely distributed as functional, inactivated, or remnant ORFs in S943 introns.     

Predicting the secondary structures and tertiary interactions of 211 group I introns in IE subgroup

The large number of currently available group I intron sequences in the public databases provides opportunity for studying this large family of structurally complex catalytic RNA by large-scale comparative sequence analysis. In this study, the detailed secondary structures of 211 group I introns in the IE subgroup were manually predicted. The secondary structure-favored alignments showed that IE introns contain 14 conserved stems. The P13 stem formed by long-range base-pairing between P2.1 and P9.1 is conserved among IE introns. Sequence variations in the conserved core divide IE introns into three distinct minor subgroups, namely IE1, IE2 and IE3. Co-variation of the peripheral structural motifs with core sequences supports that the peripheral elements function in assisting the core structure folding. Interestingly, host-specific structural motifs were found in IE2 introns inserted at S516 position. Competitive base-pairing is found to be conserved at the junctions of all long-range paired regions, suggesting a possible mechanism of establishing long-range base-pairing during large RNA folding. These findings extend our knowledge of IE introns, indicating that comparative analysis can be a very good complement for deepening our understanding of RNA structure and function in the genomic era.