The natural history of group I introns

There are four major classes of introns: self-splicing group I and group II introns, tRNA and/or archaeal introns and spliceosomal introns in nuclear pre-mRNA. Group I introns are widely distributed in protists, bacteria and bacteriophages. Group II introns are found in fungal and land plant mitochondria, algal plastids, bacteria and Archaea. Group II and spliceosomal introns share a common splicing pathway and might be related to each other. The tRNA and/or archaeal introns are found in the nuclear tRNA of eukaryotes and in archaeal tRNA, rRNA and mRNA. The mechanisms underlying the self-splicing and mobility of a few model group I introns are well understood. By contrast, the role of these highly distinct processes in the evolution of the 1500 group I introns found thus far in nature (e.g. in algae and fungi) has only recently been clarified. The explosion of new sequence data has facilitated the use of comparative methods to understand group I intron evolution in a broader context and to generate hypotheses about intron insertion, splicing and spread that can be tested experimentally.     

Structural conventions for group I introns.

Conventions for nomenclature of structural elements and a standard secondary structure representation for group I introns have been established by workers in the field. These conventions are designed to facilitate effective communication of information concerning the structure and function of these self-splicing introns.     

New RNA-mediated reactions by yeast mitochondrial group I introns.

The group I self-splicing reaction is initiated by attack of a guanosine nucleotide at the 5' splice site of intron-containing precursor RNA. When precursor RNA containing a yeast mitochondrial group I intron is incubated in vitro under conditions of self-splicing, guanosine nucleotide attack can also occur at other positions: (i) the 3' splice site, resulting in formation of a 3' exon carrying an extra added guanosine nucleotide at its 5' end; (ii) the first phosphodiester bond in precursor RNA synthesized from the SP6 bacteriophage promoter, leading to substitution of the first 5'-guanosine by a guanosine nucleotide from the reaction mixture; (iii) the first phosphodiester bond in already excised intron RNA, resulting in exchange of the 5' terminal guanosine nucleotide for a guanosine nucleotide from the reaction mixture. An identical sequence motif (5'-GAA-3') occurs at the 3' splice site, the 5' end of SP6 precursor RNA and at the 5' end of excised intron RNA. We propose that the aberrant reactions can be explained by base-pairing of the GAA sequence to the Internal Guide Sequence. We suggest that these reactions are mediated by the same catalytic centre of the intron RNA that governs the normal splicing reactions.     

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.     

The in vivo use of alternate 3'-splice sites in group I introns.

Alternative splicing of group I introns has been postulated as a possible mechanism that would ensure the translation of proteins encoded into intronic open reading frames, discontinuous with the upstream exon and lacking an initiation signal. Alternate splice sites were previously depicted according to secondary structures of several group I introns. We present here strong evidence that, in the case of Podospora anserina nad 1-i4 and cox1-i7 mitochondrial introns, alternative splicing events do occur in vivo. Indeed, by PCR experiments we have detected molecules whose sequence is precisely that expected if the predicted alternate 3'-splice sites were used.     

Origin and evolution of group I introns in cyanobacterial tRNA genes.

Many tRNA(Leu)UAA genes from plastids contain a group I intron. An intron is also inserted in the same gene at the same position in cyanobacteria, the bacterial progenitors of plastids, suggesting an ancient bacterial origin for this intron. A group I intron has also been found in the tRNA(fMet) gene of some cyanobacteria but not in plastids, suggesting a more recent origin for this intron. In this study, we investigate the phylogenetic distributions of the two introns among cyanobacteria, from the earliest branching to the more derived species. The phylogenetic distribution of the tRNA(Leu)UAA intron follows the clustering of rRNA sequences, being either absent or present in clades of closely related species, with only one exception in the Pseudanabaena group. Our data support the notion that the tRNA(Leu)UAA intron was inherited by cyanobacteria and plastids through a common ancestor. Conversely, the tRNA(fMet) intron has a sporadic distribution, implying that many gains and losses occurred during cyanobacterial evolution. Interestingly, a phylogenetic tree inferred from intronic sequences clearly separates the different tRNA introns, suggesting that each family has its own evolutionary history.     

Metal ion interaction with cosubstrate in self-splicing of group I introns.

The catalytic mechanism for self-splicing of the group I intron in the pre-mRNA from the nrdB gene in bacteriophage T4 has been investigated using 2'-amino- 2'-deoxyguanosine or guanosine as cosubstrates in the presence of Mg2+, Mn2+and Zn2+. The results show that a divalent metal ion interacts with the cosubstrate and thereby influences the efficiency of catalysis in the first step of splicing. This suggests the existence of a metal ion that catalyses the nucleophilic attack of the cosubstrate. Of particular significance is that the transesterification reactions of the first step of splicing with 2'-amino-2'-deoxyguanosine as cosubstrate are more efficient in mixtures containing either Mn2+or Zn2+together with Mg2+than with only magnesium ions present. The experiments in metal ion mixtures show that two (or more) metal ions are crucial for the self-splicing of group I introns and suggest the possibility that more than one of these have a direct catalytic role. A working model for a two-metal-ion mechanism in the transesterification steps is suggested.     

Toward predicting self-splicing and protein-facilitated splicing of group I introns

In the current era of massive discoveries of noncoding RNAs within genomes, being able to infer a function from a nucleotide sequence is of paramount interest. Although studies of individual group I introns have identified self-splicing and nonself-splicing examples, there is no overall understanding of the prevalence of self-splicing or the factors that determine it among the >2300 group I introns sequenced to date. Here, the self-splicing activities of 12 group I introns from various organisms were assayed under six reaction conditions that had been shown previously to promote RNA catalysis for different RNAs. Besides revealing that assessing self-splicing under only one condition can be misleading, this survey emphasizes that in vitro self-splicing efficiency is correlated with the GC content of the intron (>35% GC was generally conductive to self-splicing), and with the ability of the introns to form particular tertiary interactions. Addition of the Neurospora crassa CYT-18 protein activated splicing of two nonself-splicing introns, but inhibited the second step of self-splicing for two others. Together, correlations between sequence, predicted structure and splicing begin to establish rules that should facilitate our ability to predict the self-splicing activity of any group I intron from its sequence.     

Vertical evolution and intragenic spread of lichen-fungal group I introns

One family within the Euascomycetes (Ascomycota), the lichen-forming Physciaceae, is particularly rich in nuclear ribosomal [r]DNA group I introns. We used phylogenetic analyses of group I introns and lichen-fungal host cells to address four questions about group I intron evolution in lichens, and generally in all eukaryotes: 1) Is intron spread in the lichens associated with the intimate association of the fungal and photosynthetic cells that make up the lichen thallus? 2) Are the multiple group I introns in the lichen-fungi of independent origins, or have existing introns spread into novel sites in the rDNA? 3) If introns have moved to novel sites, then does the exon context of these sites provide insights into the mechanism of intron spread? and 4) What is the pattern of intron loss in the small subunit rDNA gene of lichen-fungi? Our analyses show that group I introns in the lichen-fungi and in the lichen-algae (and lichenized cyanobacteria) do not share a close evolutionary relationship, suggesting that these introns do not move between the symbionts. Many group I introns appear to have originated in the common ancestor of the Lecanorales, whereas others have spread within this lineage (particularly in the Physciaceae) putatively through reverse-splicing into novel rRNA sites. We suggest that the evolutionary history of most lichen-fungal group I introns is characterized by rare gains followed by extensive losses in descendants, resulting in a sporadic intron distribution. Detailed phylogenetic analyses of the introns and host cells are required, therefore, to distinguish this scenario from the alternative hypothesis of widespread and independent intron gains in the different lichen-fungal lineages.     

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.     

Interlocked circle formation by group I introns: structural requirements and mechanism.

Precursor RNA transcribed from the yeast mitochondrial gene coding for the large ribosomal RNA contains a group I intron that can excise itself in vitro. Apart from group I specific sequence elements the intron also contains a gene encoding a DNA endonuclease involved in intron dispersal. A precursor RNA derivative from which this gene has been removed self-splices efficiently, but due to activation of cryptic opening sites located in the 5' exon, the 3' part of this exon is sometimes co-excised with the intron. Upon further reaction, this enlarged intron molecules give rise to interlocked circles, comprising small circles derived from 5' exon parts and large circles of the intron. Sequence comparison between cryptic opening sites and authentic splice sites reveals in most cases homology with the 3' exon part that is capable of interacting with the Internal Guide Sequence. The role of the IGS was further substantiated by replacing the cryptic opening sites with well defined sequences of authentic splice sites: one corresponding to the 3' splice site and its mutant derivatives, the other to a fragment containing the natural 5'-3' exon junction. Precursor RNAs derived from these constructs give rise to interlocked circles, and mutation studies confirm that the 3' exon nucleotides flanking a 3' splice site are essential for their formation. The results underline the crucial role of the IGS in interlocked circle formation which behaves similarly as in the normal self-splicing reactions. It has been proposed that the two short helices formed by basepairing of the IGS with the 5' and 3' exon can co-axially stack on top of each other forming a quasi continuous RNA double helix or pseudoknot. We present a model explaining how transesterification reactions of a mutant precursor RNA in such a pseudoknot can lead to interlocked circles. The experiments support the notion that a similar structure is also operative in splicing of wild type precursor RNA.     

Streptomycin inhibits splicing of group I introns by competition with the guanosine substrate.

Streptomycin is an aminocyclitol glycoside antibiotic, which interferes with prokaryotic protein synthesis by interacting with the ribosomal RNA. We report here that streptomycin is also able to inhibit self splicing of the group I intron of the thymidylate synthase gene of phage T4. The inhibition is kinetically competitive with the substrate guanosine. Streptomycin and guanosine have in common a guanidino group, which has been shown to undergo hydrogen bonds with the ribozyme (Bass & Cech, Biochemistry, 25, 1986, 4473). The inhibitory effect of streptomycin extends to other group I introns, but does not affect group II introns. Mutating the bulged nucleotide in the conserved P7 secondary structure element of the td intron alters the affinity of the ribozyme for both guanosine and streptomycin. Myomycin, an antibiotic with similar effects on protein synthesis as streptomycin, is also able to inhibit splicing. In contrast, bluensomycin, which is structurally related to streptomycin, but contains only one guanidino group does not inhibit splicing. We discuss these findings in support of an evolutionary model that stresses the antiquity of antibiotics (J. Davies, Molecular Microbiology 4, 1990, 1227).     

The ins and outs of group II introns

Group II introns have attracted considerable attention as ribozymes, mobile genetic elements and possible progenitors of nuclear spliceosomal introns. Major advances in understanding their catalytic structure and dispersal strategies have recently come from several model mitochondrial and bacterial self-splicing introns. In Nature, this family of introns shows wide variation in both features and behaviour, and this review includes a focus on the diversity of evolutionary pathways taken.     

Invasion of protein coding genes by green algal ribosomal group I introns

The spread of group I introns depends on their association with intron-encoded homing endonucleases. Introns that encode functional homing endonuclease genes (HEGs) are highly invasive, whereas introns that only encode the group I ribozyme responsible for self-splicing are generally stably inherited (i.e., vertical inheritance). A number of recent case studies have provided new knowledge on the evolution of group I introns, however, there are still large gaps in understanding of their distribution on the tree of life, and how they have spread into new hosts and genic sites. During a larger phylogenetic survey of chlorophyceaen green algae, we found that 23 isolates contain at least one group I intron in the rbcL chloroplast gene. Structural analyses show that the introns belong to one of two intron lineages, group IA2 intron-HEG (GIY-YIG family) elements inserted after position 462 in the rbcL gene, and group IA1 introns inserted after position 699. The latter intron type sometimes encodes HNH homing endonucleases. The distribution of introns was analyzed on an exon phylogeny and patterns were recovered that are consistent with vertical inheritance and possible horizontal transfer. The rbcL 462 introns are thus far reported only within the Volvocales, Hydrodictyaceae and Bracteacoccus, and closely related isolates of algae differ in the presence of rbcL introns. Phylogenetic analysis of the intron conserved regions indicates that the rbcL699 and rbcL462 introns have distinct evolutionary origins. The rbcL699 introns were likely derived from ribosomal RNA L2449 introns, whereas the rbcL462 introns form a close relationship with psbA introns.     

The DNA polymerase genes of several HMU-bacteriophages have similar group I introns with highly divergent open reading frames.

A previous report described the discovery of a group I, self-splicing intron in the DNA polymerase gene of the Bacillus subtilis bacteriophage SPO1 (1). In this study, the DNA polymerase genes of three close relatives of SPO1: SP82, 2C and phi e, were also found to be interrupted by an intron. All of these introns have group I secondary structures that are extremely similar to one another in primary sequence. Each is interrupted by an open reading frame (ORF) that, unlike the intron core or exon sequences, are highly diverged. Unlike the relatives of Escherichia coli bacteriophage T4, most of which do not have introns (2), this intron seems to be common among the relatives of SPO1.