Reverse splicing of the Tetrahymena IVS: evidence for multiple reaction sites in the 23S rRNA.

Group I introns in rRNA genes are clustered in highly conserved regions that include tRNA and mRNA binding sites. This pattern is consistent with insertion of group I introns by direct interaction with exposed regions of rRNA. Integration of the Tetrahymena group I intron (or intervening sequence, IVS) into large subunit rRNA via reverse splicing was investigated using E. coli 23S rRNA as a model substrate. The results show that sequences homologous to the splice junction in Tetrahymena are the preferred site of integration, but that many other sequences in the 23S rRNA provide secondary targets. Like the original splice junction, many new reaction sites are in regions of stable secondary structure. Reaction at the natural splice junction is observed in 50S subunits and to a lesser extent in 70S ribosomes. These results support the feasibility of intron transposition to new sites in rRNA genes via reverse splicing.     

125 Changing Environmental Conditions and Changes in the Abundances of Rocky Intertidal Seaweeds on Southern California Shores

In a recent study of the North American biogeography of the red algae genus Hildenbrandia , the presence of group I introns were noted in the nuclear SSU rRNA gene of the marine species H. rubra (Hildenbrandiales). Group I introns in the nuclear encoded rRNAs have been previously reported in the Hildenbrandiales as well as the Bangiales. All reported introns within the red algae have been identified as belonging to the IC1 subclass and occur at two insertion sites in the nuclear small subunit rRNA (516 and 1506). However, an unclassified intron was discovered at position 989 in the nuclear SSU rRNA gene of a collection of H. rubra from British Columbia, Canada. We have determined that the intron is a member of the IE subclass and this is the first report of an IE intron and an intron in position 989 in the red algae. Phylogenetic analyses of the intron sequences reveal a close relationship between this group IE intron and similar ascomycete and basidiomycete fungal IE introns in the nuclear SSU rRNA genes at positions 989 and 1199. In addition, a common unique helix (structural signature) in the P13 domain of the Hildenbrandia intron and those of the fungi at the 989 and 1199 IE positions in the nuclear SSU rRNA gene also indicates a close relationship. Hence, this study provides evidence for a possible lateral transfer of the IE intron in position 989 between fungal and red algal nuclear SSU rRNA genes.     

Divergent Histories of rDNA Group I Introns in the Lichen Family Physciaceae

The wide but sporadic distribution of group I introns in protists, plants, and fungi, as well as in eubacteria, likely resulted from extensive lateral transfer followed by differential loss. The extent of horizontal transfer of group I introns can potentially be determined by examining closely related species or genera. We used a phylogenetic approach with a large data set (including 62 novel large subunit [LSU] rRNA group I introns) to study intron movement within the monophyletic lichen family Physciaceae. Our results show five cases of horizontal transfer into homologous sites between species but do not support transposition into ectopic sites. This is in contrast to previous work with Physciaceae small subunit (SSU) rDNA group I introns where strong support was found for multiple ectopic transpositions. This difference in the apparent number of ectopic intron movements between SSU and LSU rDNA genes may in part be explained by a larger number of positions in the SSU rRNA, which can support the insertion and/or retention of group I introns. In contrast, we suggest that the LSU rRNA may have fewer acceptable positions and therefore intron spread is limited in this gene. Reviewing Editor: Dr. W. Ford Doolittle     

Widespread occurrence of spliceosomal introns in the rDNA genes of ascomycetes

Spliceosomal (pre-mRNA) introns have previously been found in eukaryotic protein-coding genes, in the small nuclear RNAs of some fungi, and in the small- and large-subunit ribosomal DNA genes of a limited number of ascomycetes. How the majority of these introns originate remains an open question because few proven cases of recent and pervasive intron origin have been documented. We report here the widespread occurrence of spliceosomal introns (69 introns at 27 different sites) in the small- and large-subunit nuclear-encoded rDNA of lichen-forming and free-living members of the Ascomycota. Our analyses suggest that these spliceosomal introns are of relatively recent origin, i.e., within the Euascomycetes, and have arisen through aberrant reverse-splicing (in trans) of free pre-mRNA introns into rRNAs. The spliceosome itself, and not an external agent (e.g., transposable elements, group II introns), may have given rise to these introns. A nonrandom sequence pattern was found at sites flanking the rRNA spliceosomal introns. This pattern (AG-intron-G) closely resembles the proto-splice site (MAG-intron-R) postulated for intron insertions in pre-mRNA genes. The clustered positions of spliceosomal introns on secondary structures suggest that particular rRNA regions are preferred sites for insertion through reverse-splicing.     

124 Evidence for Lateral Transfer of a IE Intron between Fungal and Red Algal Small Subunit Ribosomal RNA Genes

In a recent study of the North American biogeography of the red algae genus Hildenbrandia, the presence of group I introns were noted in the nuclear SSU rRNA gene of the marine species H. rubra (Hildenbrandiales). Group I introns in the nuclear encoded rRNAs have been previously reported in the Hildenbrandiales as well as the Bangiales. All reported introns within the red algae have been identified as belonging to the IC1 subclass and occur at two insertion sites in the nuclear small subunit rRNA (516 and 1506). However, an unclassified intron was discovered at position 989 in the nuclear SSU rRNA gene of a collection of H. rubra from British Columbia, Canada. We have determined that the intron is a member of the IE subclass and this is the first report of an IE intron and an intron in position 989 in the red algae. Phylogenetic analyses of the intron sequences reveal a close relationship between this group IE intron and similar ascomycete and basidiomycete fungal IE introns in the nuclear SSU rRNA genes at positions 989 and 1199. In addition, a common unique helix (structural signature) in the P13 domain of the Hildenbrandia intron and those of the fungi at the 989 and 1199 IE positions in the nuclear SSU rRNA gene also indicates a close relationship. Hence, this study provides evidence for a possible lateral transfer of the IE intron in position 989 between fungal and red algal nuclear SSU rRNA 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.     

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.     

The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing.     

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.     

Identification of a family of group II introns encoding LAGLIDADG ORFs typical of group I introns

Group I and group II introns are unrelated classes of introns that each encode proteins that facilitate intron splicing and intron mobility. Here we describe a new subfamily of nine introns in fungi that are group II introns but encode LAGLIDADG ORFs typical of group I introns. The introns have fairly standard group IIB1 RNA structures and are inserted into three different sites in SSU and LSU rRNA genes. Therefore, introns should not be assumed to be group I introns based solely on the presence of a LAGLIDADG ORF.     

Origin and Inheritance of Group I Introns in 26S rRNA Genes of Gaeumannomyces graminis

Studies of the distribution of the three group I introns (intron A, intron T, and intron AT) in the 26S rDNA of Gaeumannomyces graminis had suggested that they were transferred to a common ancestor of G. graminis var. avenae and var. tritici after it had branched off from var. graminis. Intron AT and intron A exhibited vertical inheritance and coevolved in concert with their hosts. Intron loss could occur after its acquisition. Loss of any one of the three introns could occur in var. tritici whereas only loss of intron T had been found in the majority of var. avenae isolates. The existence of isolates of var. tritici and var. avenae with three introns suggested that intron loss could be reversed by intron acquisition and that the whole process is a dynamic one. This process of intron acquisition and intron loss reached different equilibrium points for different varieties and subgroups, which explained the irregular distribution of these introns in G. graminis. Each of the three group I introns was more closely related to other intron sequences that share the same insertion point in the 26S rDNA than to each other. These introns in distantly related organisms appeared to have a common ancestry. This system had provided a good model for studies on both the lateral transfer and common ancestry of group I introns in the 26S rRNA genes. Received: 17 May 1996 / Accepted: 14 January 1997     

Nucleotide sequence and intron structure of the apocytochrome b gene of Neurospora crassa mitochondria

The sequence of the apocytochrome b (cob) gene of Neurospora crassa has been determined. The structural gene is interrupted by two intervening sequences of approximately 1260 bp each. The polypeptide encoded by the exons shows extensive homology with the cob proteins of Aspergillus nidulans and Saccharomyces cerevisiae (79% and 60%, respectively). The two introns are, however, located at sites different from those of introns in the cob genes of A. nidulans and S. cerevisiae (which contain highly homologous introns at the same site within the gene). The introns share several short regions of sequence homology (10-12 bp long) with each other and with other fungal mitochondrial introns. Moreover, the second intron contains a 50 nucleotide long sequence that is highly homologous with sequences within every ribosomal intron of fungal mitochondria sequenced to date. The conserved sequences may allow the formation of a core secondary structure, which is nearly identical in many mitochondrial introns. The conserved secondary structure may be required for intron splicing. The second intron contains an open reading frame, continuous with the preceding exon, of approximately 290 codons. Two stretches of 10 amino acid residues, conserved in many introns, are present in the open reading frame.     

DNA sequence and secondary structures of the large subunit rRNA coding regions and its two class I introns of mitochondrial DNA fromPodospora anserina

Summary DNA sequence analysis has shown that the gene coding for the mitochondrial (mt) large subunit ribosomal RNA (rRNA) fromPodospora anserina is interrupted by two class I introns. The coding region for the large subunit rRNA itself is 3715 bp and the two introns are 1544 (r1) and 2404 (r2) bp in length. Secondary structure models for the large subunit rRNA were constructed and compared with the equivalent structure fromEscherichia coli 23S rRNA. The two structures were remarkably similar despite an 800-base difference in length. The additional bases in theP. anserina rRNA appear to be mostly in unstructured regions in the 3 part of the RNA. Secondary structure models for the two introns show striking similarities with each other as well as with the intron models from the equivalent introns inSaccharomyces cerevisiae, Neurospora crassa, andAspergillus nidulans. The long open reading frames in each intron are different from each other, however, and the nucleotide sequence similarity diverges as it proceeds away from the core structure. Each intron is located within regions of the large subunit rRNA gene that are highly conserved in both sequence and structure. Computer analysis showed that the open reading frame for intron r1 contained a common maturase-like polypeptide. The open reading frames of intron r2 apeared to be chimeric, displaying high sequence similarity with the open reading frames in the r1 and ATPase 6 introns ofN. crassa.     

Three variant introns of the same general class in the mitochondrial gene for cytochrome oxidase subunit 1 in Aspergillus nidulans

The oxiA gene of Aspergillus nidulans, coding for cytochrome oxidase subunit 1, is shown by DNA sequencing to contain three introns. An AUG start codon is not present at the beginning of the sequence, suggesting that either another codon, possibly the four base codon AUGA, is used for initiation or there is a further short intron between the true start codon and the beginning of the recognisable coding region. The second and third introns have long open reading frames, which could code for maturase proteins. The lack of conservation of amino acid sequence in the putative region of proteolytic cleavage for maturase formation suggests that the first conserved decapeptide may act as the recognition signal for protein processing. The third intron is remarkably (70%) homologous to the second intron of the cytochrome oxidase subunit 1 gene of Schizosaccharomyces pombe and both are located in exactly the same position. The third Aspergillus intron has an in-frame insertion of a 37-bp GC-rich DNA sequence which is now flanked by a 5-bp repeat, a well-known feature of transposable elements. All three introns in the oxiA gene have a 'core' RNA secondary structure found in a class of introns fitting the RNA splicing model of Davies et al. (1982). This core RNA structure may play a catalytic as well as a structural role in intron splicing. A sequence within the intron could act as a guide to align the splice sites of two of the introns in accordance with the model of Davies et al.     

Structure of the ribosome-associated 5.8 S ribosomal RNA

The structure of the 5.8 S ribosomal RNA in rat liver ribosomes was probed by comparing dimethyl sulfate-reactive sites in whole ribosomes, 60 S subunits, the 5.8 S-28 S rRNA complex and the free 5.8 S rRNA under conditions of salt and temperature that permit protein synthesis in vitro. Differences in reactive sites between the free and both the 28 S rRNA and 60 S subunit-associated 5.8 S rRNA show that significant conformational changes occur when the molecule interacts with its cognate 28 S rRNA and as the complex is further integrated into the ribosomal structure. These results indicate that, as previously suggested by phylogenetic comparisons of the secondary structure, only the "G + C-rich" stem may remain unaltered and a universal structure is probably present only in the whole ribosome or 60 S subunit. Further comparisons with the ribosome-associated molecule indicate that while the 5.8 S rRNA may be partly localized in the ribosomal interface, four cytidylic acid residues, C56, C100, C127 and C128, remain reactive even in whole ribosomes. In contrast, the cytidylic acid residues in the 5 S rRNA are not accessible in either the 60 S subunit or the intact ribosome. The nature of the structural rearrangements and potential sites of interaction with the 28 S rRNA and ribosomal proteins are discussed.     

Long-term evolution of the S788 fungal nuclear small subunit rRNA group I introns

More than 1000 group I introns have been identified in fungal rDNA. Little is known, however, of the splicing and secondary structure evolution of these ribozymes. Here, we use a combination of comparative and biochemical methods to address the evolution and splicing of a vertically inherited group I intron found at position 788 in the fungal small subunit (S) rRNA. The ancestral state of the S788 intron contains a highly conserved core and an extended P5 domain typical of IC1 introns. In contrast, the more derived introns have lost most of P5, and have an accelerated divergence rate within the core region with three functionally important substitutions that unambiguously separate them from the ancestral pool. Of 14 S788 group I introns that were tested for splicing, five, all of the ancestral type, were able to self-splice and produced intron RNA circles in vitro. The more derived S788 introns did not self-splice, and potentially rely on fungal-specific factors to facilitate splicing. In summary, we demonstrate one possible fate of vertically inherited group I introns, the loss of secondary structure elements, lessened selective constraints in the intron core, and ultimately, dependence on host-mediated splicing.