Intron 5 alpha of the COXI gene of yeast mitochondrial DNA is a mobile group I intron.

We have found that intron 5 alpha of the COXI gene (al5 alpha) of yeast mtDNA is a mobile group I intron in crosses between strains having or lacking the intron. We have demonstrated the following hallmarks of that process: 1) co-conversion of flanking optional intron markers; 2) mutations that truncate the intron open reading frame block intron mobility; and 3) the intron open reading frame encodes an endonuclease activity that is required for intron movement. The endonuclease activity, termed I-Sce IV, cleaves the COXI allele lacking al5 alpha near the site of intron insertion, making a four-base staggered cut with 3' OH overhangs. Three cloned DNAs derived from different forms of the COXI gene, which differ in primary sequence at up to seven nucleotides around the cleavage site, are all good substrates for in vitro I-Sce IV cleavage activity. Two of the strains from which these substrates were derived were tested in crosses and are comparably efficient as al5 alpha recipients. When compared with omega mobility occurring simultaneously in one cross, al5 alpha is less efficient as a mobile element.     

Abortive transposition by a group II intron in yeast mitochondria

Group II intron homing in yeast mitochondria is initiated at active target sites by activities of intron-encoded ribonucleoprotein (RNP) particles, but is completed by competing recombination and repair mechanisms. Intron aI1 transposes in haploid cells at low frequency to target sites in mtDNA that resemble the exon 1-exon 2 (E1/E2) homing site. This study investigates a system in which aI1 can transpose in crosses (i.e., in trans). Surprisingly, replacing an inefficient transposition site with an active E1/E2 site supports <1% transposition of aI1. Instead, the ectopic site was mainly converted to the related sequence in donor mtDNA in a process we call "abortive transposition." Efficient abortive events depend on sequences in both E1 and E2, suggesting that most events result from cleavage of the target site by the intron RNP particles, gapping, and recombinational repair using homologous sequences in donor mtDNA. A donor strain that lacks RT activity carries out little abortive transposition, indicating that cDNA synthesis actually promotes abortive events. We also infer that some intermediates abort by ejecting the intron RNA from the DNA target by forward splicing. These experiments provide new insights to group II intron transposition and homing mechanisms in yeast mitochondria.     

DNA polymerization catalysed by a group II intron RNA in vitro.

The excised group II intron bI1 from Saccharomyces cerevisiae can act as a ribozyme catalysing various chemical reactions with different substrate RNAs in vitro . Recently, we have described an editing-like RNA polymerization reaction catalysed by the bI1 intron lariat that proceeds in the 3'-->5'direction. Here we show that the bI1 lariat RNA can also catalyse successive deoxyribonucleotide polymerization reactions on exogenous substrate molecules. The basic mechanism of the reaction involved interacting cycles between an alternative version of partial reverse splicing (lariat charging) and canonical forward splicing (lariat discharging by exon ligation). With an overall chain growth in the 3'-->5' direction, the 5' exon RNAs (IBS1dN) were elongated by successive insertion of deoxyribonucleotides derived from single deoxyribonucleotide substitutions (dA, dG, dC or dT). All four deoxyribonucleotides were used as substrates, although with different efficiencies. Our findings extend the catalytic repertoire of group II intron RNAs not only by a novel DNA polymerization activity, but also by a DNA-DNA ligation capacity, supporting the idea that ribozymes might have been part of the first primordial polymerization machinery for both RNA and DNA.     

Interlocked RNA circle formation by a self-splicing yeast mitochondrial group I intron

RNA containing the aI3 group I intron of the yeast mitochondrial gene encoding cytochrome oxidase subunit I shows self-splicing in vitro. The excised intron, comprising 1514 nucleotides, is partially split into an upstream portion, containing the intronic reading frame, and a downstream portion, containing the typical group I conserved sequence elements. Full-length intron RNA and intron parts occur in linear and circular form. In the transesterification reactions leading to circle formation, only the guanosine nucleotide added during splicing is removed. Reincubation of isolated, complete circular intron RNA under self-splicing conditions leads to formation of free subintronic RNA circles. Under similar conditions, purified linear intron RNA gives rise to a number of circular and linear products, one of which consists of interlocked subintronic RNA circles. These observations suggest that the intron RNA possesses a dynamic structure in which subtle alterations in folding result in the formation of RNA products with different topology.     

Homing of a group II intron from Lactococcus lactis subsp. lactis ML3.

Ll.ltrB is a functional group II intron located within a gene (ltrB) encoding a conjugative relaxase essential for transfer of the lactococcal element pRSO1. In this work, the Ll.ltrB intron was shown to be an independent mobile element capable of inserting into an intronless allele of the ltrB gene. Ll.ltrB was not observed to insert into a deletion derivative of the ltrB gene in which the intron splice site was removed. In contrast, a second vector containing a 271-nucleotide segment of ltrB spanning the Ll.ltrB splice site was shown to be a proficient recipient of intron insertion. Efficient homing was observed in the absence of a functional host homologous recombination system. This work demonstrates that the Ll.ltrB intron is a novel site-specific mobile element in lactococci and that group II intron self-transfer is a mechanism for intron dissemination among bacteria.     

CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron.

The terminal intron (bI2) of the yeast mitochondrial cytochrome b gene is a group I intron capable of self-splicing in vitro at high concentrations of Mg2+. Excision of bI2 in vivo, however, requires a protein encoded by the nuclear gene CBP2. The CBP2 protein has been partially purified from wild-type yeast mitochondria and shown to promote splicing at physiological concentrations of Mg2+. The self-splicing and protein-dependent splicing reactions utilized a guanosine nucleoside cofactor, the hallmark of group I intron self-splicing reactions. Furthermore, mutations that abolished the autocatalytic activity of bI2 also blocked protein-dependent splicing. These results indicated that protein-dependent excision of bI2 is an RNA-catalyzed process involving the same two-step transesterification mechanism responsible for self-splicing of group I introns. We propose that the CBP2 protein binds to the bI2 precursor, thereby stabilizing the catalytically active structure of the RNA. The same or a similar RNA structure is probably induced by high concentrations of Mg2+ in the absence of protein. Binding of the CBP2 protein to the unspliced precursor was supported by the observation that the protein-dependent reaction was saturable by the wild-type precursor. Protein-dependent splicing was competitively inhibited by excised bI2 and by a splicing-defective precursor with a mutation in the 5' exon near the splice site but not by a splicing-defective precursor with a mutation in the core structure. Binding of the CBP2 protein to the precursor RNA had an effect on the 5' splice site helix, as evidenced by suppression of the interaction of an exogenous dinucleotide with the internal guide sequence.     

Extensive mis-splicing of a bi-partite plant mitochondrial group II intron

Expression of the seed plant mitochondrial nad5 gene involves two trans-splicing events that remove fragmented group II introns and join the small, central exon c to exons b and d. We show that in both monocot and eudicot plants, extensive mis-splicing of the bi-partite intron 2 takes place, resulting in the formation of aberrantly spliced products in which exon c is joined to various sites within exon b. These mis-spliced products accumulate to levels comparable to or greater than that of the correctly spliced mRNA. We suggest that mis-splicing may result from folding constraints imposed on intron 2 by base-pairing between exon a and a portion of the bi-partite intron 3 downstream of exon c. Consistent with this hypothesis, we find that mis-splicing does not occur in Oenothera mitochondria, where intron 3 is further fragmented such that the predicted base-pairing region is not covalently linked to exon c. Our findings suggest that intron fragmentation may lead to mis-splicing, which may be corrected by further intron fragmentation.     

Structure-function relationships in a self-splicing group II intron: a large part of domain II of the mitochondrial intron aI5 is not essential for self-splicing.

An oligonucleotide-directed deletion of 156 nucleotides has been introduced into the yeast mitochondrial group II intron al5 (887 nt). The deletion comprises almost all of domain II, which is one of the six phylogenetically conserved structural elements of group II introns. This mutant displays reduced self-splicing activity, but results of chemical probing with dimethylsulphate suggest that sequences at the site of the deletion interfere with the normal folding of the intron. This is supported by computer analyses, which predict a number of alternative structures involving conserved intron sequences. Splicing activity could be restored by insertion of a 10-nucleotide palindromic sequence into the unique Smal site of the deletion mutant, resulting in the formation of a small stable stem-loop element at the position of domain II. These results provide a direct correlation between folding of the RNA and its activity. We conclude that at least a large part of domain II of the group II intron al5 is not required for self-splicing activity. This deletion mutant with a length of 731 nucleotides represents the smallest self-splicing group II intron so far known.     

Control of branch-site choice by a group II intron.

The branch site of group II introns is typically a bulged adenosine near the 3'-end of intron domain 6. The branch site is chosen with extraordinarily high fidelity, even when the adenosine is mutated to other bases or if the typically bulged adenosine is paired. Given these facts, it has been difficult to discern the mechanism by which the proper branch site is chosen. In order to dissect the determinants for branch-point recognition, new mutations were introduced in the vicinity of the branch site and surrounding domains. Single mutations did not alter the high fidelity for proper branch-site selection. However, several combinations of mutations moved the branch site systematically to new positions along the domain 6 stem. Analysis of those mutants, together with a new alignment of domain 5 and domain 6 sequences, reveals a set of structural determinants that appear to govern branch-site selection by group II introns.     

Homing of a bacterial group II intron with an intron-encoded protein lacking a recognizable endonuclease domain

RmInt1 is a functional group II intron found in Sinorhizobium meliloti where it interrupts a group of IS elements of the IS630-Tc1 family. In contrast to many other group II introns, the intron-encoded protein (IEP) of RmInt1 lacks the characteristic conserved part of the Zn domain associated with the IEP endonuclease activity. Nevertheless, in this study, we show that RmInt1 is capable of inserting into a vector containing the DNA spanning the RmInt1 target site from the genome of S. meliloti. Efficient homing was also observed in the absence of homologous recombination (RecA- strains). In addition, it is shown that RmInt1 is able to move to its target in a heterologous host (S. medicae). Homing of RmInt1 occurs very efficiently upon DNA target uptake (conjugation/electroporation) by the host cell resulting in a proportion of invaded target of 11-30%. Afterwards, the remaining intronless target DNA is protected from intron invasion.     

Promiscuous mitochondrial group II intron sequences in plant nuclear genomes

Gene translocations from the organelles to the nucleus are postulated by the endosymbiont hypothesis. We here report evidence for sequence insertions in the nuclear genomes of plants that are derived from noncoding regions of the mitochondrial genome. Fragments of mitochondrial group II introns are identified in the nuclear genomes of tobacco and a bean species. The duplicated intron sequences of 75–140 bp are derived from cis- and trans-splicing introns of genes encoding subunits 1 and 5 of the NADH dehydrogenase. The mitochondrial sequences are inserted in the vicinities of a lectin gene, different glucanase genes and a gene encoding a subunit of photosystem II. Sequence similarities between the nuclear and mitochondrial copies are in the range of 80 to 97%, suggesting recent transfer events that occurred in the basic glucanase genes before and in the lectin gene after the gene duplications in the evolution of the nuclear gene families. Overlapping regions of the same introns are in two instances also involved in intramitochondrial sequence duplications. Correspondence to: V. Knoop     

Antagonistic substrate binding by a group II intron ribozyme.

In this study, the thermodynamic properties of substrate-ribozyme recognition were explored using a system derived from group II intron ai5gamma. Substrate recognition by group II intron ribozymes is of interest because any nucleic ac?id sequence can be targeted, the recognition sequence can be quite long (>/=13 bp), and reaction can proceed with a very high degree of sequence specificity. Group II introns target their substrates throug?h the formation of base-pairing interactions with two regions of the intron (EBS1 and EBS2), which are usually located far apart in the secondary structure. These structures pair with adjacent, corresponding sites (IBS1 and IBS2) on the substrate. In order to understand the relative energetic contribution of each base-pairing interaction (EBS1-IBS1 or EBS2-IBS2) to substrate binding energy, the free energy of each helix was measured. The individual helices were found to have base-pairing free energies similar to those calculated for regular RNA duplexes of the same sequence, suggesting that each recognition helix derives its binding energy from base-pairing interactions alone and that each helix can form independently. Most interestingly, it was found that the sum of the measured individual free energies (approximately 20 kcal/mol) was much higher than the known free energy for substrate binding (approximately 12 kcal/mol). This indicates that certain group II intron ribozymes can bind their substrates in an antagonistic fashion, paying a net energetic penalty upon binding the full-length substrate. This loss of binding energy is not due to weakening of individual helices, but appears to be linked to ribozyme conformational changes induced by substrate binding. This coupling between substrate binding and ribozyme conformational rearrangement may provide a mechanism for lowering overall substrate binding energy while retaining the full information content of 13 bp, thus resulting in a mechanism for ensuring sequence specificity.     

Splicing defective mutants of the COXI gene of yeast mitochondrial DNA: initial definition of the maturase domain of the group II intron aI2.

Six mutations blocking the function of a seven intron form of the mitochondrial gene encoding subunit I of cytochrome c oxidase (COXI) and mapping upstream of exon 3 were isolated and characterized. A cis-dominant mutant of the group IIA intron 1 defines a helical portion of the C1 substructure of domain 1 as essential for splicing. A trans-recessive mutant confirms that the intron 1 reading frame encodes a maturase function. A cis-dominant mutant in exon 2 was found to have no effect on the splicing of intron 1 or 2. A trans-recessive mutant, located in the group IIA intron 2, demonstrates for the first time that intron 2 encodes a maturase. A genetic dissection of the five missense mutations present in the intron 2 reading frame of that strain demonstrates that the maturase defect results from one or both of the missense mutations in a newly-recognized conserved sequence called domain X.     

Self-splicing of a group II intron in yeast mitochondria: dependence on 5'' exon sequences.

It has been previously suggested that self-splicing of group II introns starts with a nucleophilic attack of the 2' OH group from the branchpoint adenosine on the 5' splice junction. To investigate the sequences governing the specificity of this attack, a series of Bal31 nuclease deletion mutants was constructed in which progressively larger amounts of 5' exon have been removed starting from its 5' end. The ability of mutant RNAs to carry out self-splicing in vitro was studied. Involvement of 5' exon sequences in self-splicing activity is indicated by the fact that a mutant in which as many as 18 nucleotides of 5' exon remain is seriously disturbed in splicing, while larger deletions eliminate splicing entirely. Mutants containing a truncated 5' exon form aberrant RNAs. One of these is a 425-nucleotide RNA containing the 5' exon as well as sequences of the 5' part of the intron. Its 3' end maps at position 374 of the 887-nucleotide intron. The other is a less abundant lariat RNA probably originating from the remainder of the intron linked to the 3' exon. We interpret this large dependence of reactivity of the intron on 5' exon and adjoining intron sequences as evidence for base-pairing interactions between the exon and parts of the intron, leading to an RNA folding necessary for splicing. Possible folding models are discussed.