Intron Mobility Research Articles

Population Dynamics of Archaeal Mobile Introns in Natural Environments: A Shrewd Invasion Strategy of the Latent Parasitic DNA.

A body of data has been amassed in recent years, which shows the existence of introns within rRNA gene loci of certain archaeal lineages. Most striking is that the introns are optional, present in some strains and absent in others. They often have a nested gene encoding a LAGLIDADG-type site-specific DNA endonuclease known as endonuclease (HE). Such introns are, therefore, assumed to be which propagate horizontally by using the homing pathway. The phenomenon whereby mobile introns are inserted into the archaeal rRNA gene loci and disperse within a population to create genome polymorphism can be interpreted as selfish behavior of HE genes. The intron-encoded HE does not break rRNA genes containing the introns, but acts selectively to break only the intronless alleles. In other words, HE destroys alleles which do not possess internally a copy of its own gene by introducing a double strand DNA break (DSB), thus endangering the continued existence of the host's genome in that failure to repair the DSB will prove lethal. Moreover, in making its own gene into a mould for the purpose of recombination, it is adopting the horizontal propagation strategy to increase the copy number of its own gene within the population. HE is a vital factor in the propagation of this type of mobile intron, and it is a noteworthy fact that the HE gene is neutral, being neither advantageous nor disadvantageous to the existence of the host. Since the HE gene is removed along with the intron in the splicing process, it has no effect on the functional expression of the rRNA gene. Here, we discuss the dynamics whereby these molecular parasites, which reside in the archaeal genomes, propagate and evolve within a population.

Homing endonuclease and mobile intron in the novel hyperthermophilic archaeon <i>Aeropyrum pernix</i> isolated from marine hydrothermal vent

Homing endonuclease and mobile intron in the novel hyperthermophilic archaeon <i>Aeropyrum pernix</i> isolated from marine hydrothermal vent

Mutations in the Lactococcus lactis Ll.LtrB group II intron that retain mobility in vivo

BackgroundGroup II introns are mobile genetic elements that form conserved secondary and tertiary structures. In order to determine which of the conserved structural elements are required for mobility, a series of domain and sub-domain deletions were made in the Lactococcus lactis group II intron (Ll.LtrB) and tested for mobility in a genetic assay. Point mutations in domains V and VI were also tested.ResultsThe largest deletion that could be made without severely compromising mobility was 158 nucleotides in DIVb(1–2). This mutant had a mobility frequency comparable to the wild-type Ll.LtrB intron (ΔORF construct). Hence, all subsequent mutations were done in this mutant background. Deletion of DIIb reduced mobility to approximately 18% of wild-type, while another deletion in domain II (nts 404–459) was mobile to a minor extent. Only two deletions in DI and none in DIII were tolerated. Some mobility was also observed for a DIVa deletion mutant. Of the three point mutants at position G3 in DV, only G3A retained mobility. In DVI, deletion of the branch-point nucleotide abolished mobility, but the presence of any nucleotide at the branch-point position restored mobility to some extent.ConclusionsThe smallest intron capable of efficient retrohoming was 725 nucleotides, comprising the DIVb(1–2) and DII(ii)a,b deletions. The tertiary elements found to be nonessential for mobility were alpha, kappa and eta. In DV, only the G3A mutant was mobile. A branch-point residue is required for intron mobility.

Productive folding to the native state by a group II intron ribozyme

Group II introns are large catalytic RNA molecules that fold into compact structures essential for the catalysis of splicing and intron mobility reactions. Despite a growing body of information on the folded state of group II introns at equilibrium, there is currently no information on the folding pathway and little information on the ionic requirements for folding. Folding isotherms were determined by hydroxyl radical footprinting for the 32 individual protections that are distributed throughout a group II intron ribozyme derived from intron ai5γ. The isotherms span a similar range of Mg 2+ concentrations and share a similar index of cooperativity. Time-resolved hydroxyl radical footprinting studies show that all regions of the ribozyme fold slowly and with remarkable synchrony into a single catalytically active structure at a rate comparable to those of other ribozymes studied thus far. The rate constants for the formation of tertiary contacts and recovery of catalytic activity are identical within experimental error. Catalytic activity analyses in the presence of urea provide no evidence that the slow folding of the ai5γ intron is attributable to the presence of unproductive kinetic traps along the folding pathway. Taken together, the data suggest that the rate-limiting step for folding of group II intron ai5γ occurs early along the reaction pathway. We propose that this behavior resembles protein folding that is limited in rate by high contact order, or the need to form key tertiary interactions from partners that are located far apart in the primary or secondary structure.

Kinetic dissection of the multistep process in L1.ltrB intron mobility.

A kinetic characterization is presented for intron insertion into a target duplex DNA site during L1.ltrB intron mobility. This reaction is catalyzed by a ribonucleoprotein particle (RNP), consisting of a lariat form of group II intron RNA and the intron-encoded LtrA protein. In the first stage of intron mobility, the RNA component of the enzyme by itself inserts directly into the target ds DNA site, and thus, the RNP enzyme does not carry out turnover. Using single-turnover kinetics, we established an in vitro kinetic assay system and investigated mechanism of the intron RNA insertion process.

Degeneration of a homing endonuclease and its target sequence in a wild yeast strain.

Mobile introns and inteins self-propagate by 'homing', a gene conversion process initiated by site-specific homing endonucleases. The VMA intein, which encodes the PI-SceI endonuclease in Saccharomyces cerevisiae, is present in several different yeast strains. Surprisingly, a wild wine yeast (DH1-1A) contains not only the intein(+) allele, but also an inteinless allele that has not undergone gene conversion. To elucidate how these two alleles co-exist, we characterized the endonuclease encoded by the DH1-1A intein(+) allele and the target site in the intein(-) allele. Sequence analysis reveals seven mutations in the 31 bp recognition sequence, none of which occurs at positions that are individually critical for activity. However, binding and cleavage of the sequence by PI-SceI is reduced 10-fold compared to the S.cerevisiae target. The PI-SceI analog encoded by the DH1-1A intein(+) allele contains 11 mutations at residues in the endonuclease and protein splicing domains. None affects protein splicing, but one, a R417Q substitution, accounts for most of the decrease in DNA cleavage and DNA binding activity of the DH1-1A protein. Loss of activity in the DH1-1A endonuclease and target site provides one explanation for co-existence of the intein(+) and intein(-) alleles.

Intron Mobility Results in Rearrangement in Mitochondrial DNAs of Heterokaryon Incompatible Aspergillus japonicus Strains after Protoplast Fusion

Mitochondrial transmission was carried out under selective conditions between incompatible Aspergillus japonicus strains always using an oligomycin-resistant mitochondrial donor and selecting for recipient nuclei and oligomycin-resistant mitochondria. All attempted intraspecific mitochondrial transmissions were successful, but the transmission between closely related A. japonicus and A. aculeatus failed. Under selection pressure, resistant progeny harbor the mitochondrial DNA (mtDNA) of the donor strain, which may remain unchanged or may be modified by the introns of the recipient mitochondrial genome. Detailed analysis of a certain strain harboring rearranged mtDNA suggests that the mtDNA profiles of recombinant-like progeny are strongly influenced by the characteristics and mobility of introns of both parental mtDNAs. Both intron loss and intron acquisition play a role in the rearrangement of mtDNA. In certain parental combinations, a particular intron was lost very frequently.

Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference

Group II intron mobility occurs by a target DNA-primed reverse transcription mechanism in which the intron RNA reverse splices directly into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it as a primer to reverse transcribe the inserted intron RNA. The group II intron endonuclease, which mediates this process, is an RNP particle that contains the intron-encoded protein and the excised intron RNA and uses both cooperatively to recognize DNA target sequences. Here, we analyzed the interaction of the Lactococcus lactis Ll.LtrB group II intron endonuclease with its DNA target site by DNA footprinting and modification-interference approaches. In agreement with previous mutagenesis experiments showing a relatively large target site, DNase I protection extends from position −25 to +19 from the intron-insertion site on the top strand and from −28 to +16 on the bottom strand. Our results suggest that the protein first recognizes a small number of specific bases in the distal 5′-exon region of the DNA target site via major-groove interactions. These base interactions together with additional phosphodiester-backbone interactions along one face of the helix promote DNA unwinding, enabling the intron RNA to base-pair to DNA top-strand positions −12 to +3 for reverse splicing. Notably, DNA unwinding extends to at least position +6, somewhat beyond the region that base-pairs with the intron RNA, but is not dependent on interaction of the conserved endonuclease domain with the 3′ exon. Bottom-strand cleavage occurs after reverse splicing and requires recognition of a small number of additional bases in the 3′ exon, the most critical being T+5 in the now single-stranded downstream region of the target site. Our results provide the first detailed view of the interaction of a group II intron endonuclease with its DNA target site.

TESTING THE REVERSE‐SPLICING MODEL OF RDNA INTRON ORIGIN

Bhattacharya, D., Simon, D., Chakravarty, S. & Huang, J.Department of Biological Sciences and Interdepartmental Genetics Program, University of Iowa, Iowa City, IA 52242 USAGroup I introns are widely distributed in the nuclear ribosomal RNA genes of algae and fungi. Two models are proposed for intron mobility. The first relies on an endonuclease open reading frame that allows intron “homing” at the DNA‐level into homologous allelic sites. The second model proposes mobility at the RNA‐level through reverse‐splicing. Whereas endonuclease‐mediated homing is depen‐dent on a significant sequence requirement (14‐35 nt) at the insertion site, reverse‐splicing requires as little as 4‐6 nt at the 5′ flanking sequence to initiate intron integration. For this reason, reverse‐splicing may play the more important role in intron lateral transfer in the nuclear genome. In this presentation, we test two major predictions of the reverse‐splicing model with introns in the small (SSU) and large subunit (LSU) nuclear rRNA: 1) Group I introns reverse‐splice into rRNA regions which contain a 4‐6 nt 5′‐exon flanking sequence that builds a helix with the intron internal guide sequence required for forward‐ and reverse‐splicing. 2) Introns are non‐randomly distributed, with most of them clustering in regions that are not “hidden” by rRNA tertiary structure. Phylogenetic analyses show that multiple group I introns in the SSU rDNA of lichen fungi have likely originated through reverse‐splicing of one intron into a heterologous site. The reverse‐splicing model is also supported by the observation that both group I and spliceosomal introns are primarily found in highly conserved rRNA regions that map on the surface of the SSU ribosome. These introns show a positive correlation with rRNA sites that form cross‐links with tRNA and mRNA.

A novel group I intron-encoded endonuclease specific for the anticodon region of tRNAfMet genes

Open reading frames (ORFs) are frequently inserted into group I self-splicing introns. These ORFs encode either maturases that are required for splicing of the intron or DNA endonucleases that promote intron mobility. A self-splicing intron in the tRNAfMet gene of Synechocystis PCC 6803, which has been proposed to have moved laterally within the cyanobacteria, contains an ORF that is unrelated to known intron-encoded endonucleases or maturases. Here, using an in vitro transcription–translation system, we show that this intronic ORF encodes a double-strand DNA endonuclease, I-Ssp6803I. I-Ssp6803I cleaves each strand of the intronless tRNAfMet gene adjacent to the anticodon triplet leaving 3 bp 3′ extensions and has no activity at intron–exon boundaries. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a partially palindromic 20 bp region encompassing the entire anticodon stem and loop of the tRNAfMet gene. I-Ssp6803I represents a novel intron-encoded DNA endonuclease and is the first example of a chromosomally encoded group I intron endonuclease in bacteria.

A novel group I intron‐encoded endonuclease specific for the anticodon region of tRNAfMet genes

Open reading frames (ORFs) are frequently inserted into group I self-splicing introns. These ORFs encode either maturases that are required for splicing of the intron or DNA endonucleases that promote intron mobility. A self-splicing intron in the tRNA(fMet) gene of Synechocystis PCC 6803, which has been proposed to have moved laterally within the cyanobacteria, contains an ORF that is unrelated to known intron-encoded endonucleases or maturases. Here, using an in vitro transcription-translation system, we show that this intronic ORF encodes a double-strand DNA endonuclease, I-Ssp6803I. I-Ssp6803I cleaves each strand of the intronless tRNA(fMet) gene adjacent to the anticodon triplet leaving 3 bp 3' extensions and has no activity at intron-exon boundaries. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a partially palindromic 20 bp region encompassing the entire anticodon stem and loop of the tRNA(fMet) gene. I-Ssp6803I represents a novel intron-encoded DNA endonuclease and is the first example of a chromosomally encoded group I intron endonuclease in bacteria.

Intragenic suppressors that restore the splicing and homing activities of the protein encoded by the second intron of the Saccharomyces capensis cyt b gene

The second (bi2) intron of the mitochondrial cyt b gene from Saccharomyces capensis encodes a bifunctional protein which acts both as a maturase, promoting intron splicing, and as a homing-endonuclease, I-ScaI, promoting intron mobility. In this work we isolated and characterized revertants from a respiratory-deficient mutant in which both functions of the protein have been lost. Intragenic revertants resulted mainly from monosubstitutions in the mutated codon and in one case from a distant second site mutation. All novel variants of the S. capensis bi2 intron-encoded protein are competent for the maturase activity but only two of them can partially complement the homing function.

Critical base substitutions that affect the splicing and/or homing activities of the group I intron bi2 of yeast mitochondria.

The second intron (bi2) of the cyt b gene from related Saccharomyces species has an extraordinarily conserved sequence and can have different functions in wild-type cells. The protein encoded by the S. cerevisiae intron functions as a maturase to promote intron splicing, while the homologous S. capensis intron encodes a bifunctional protein that acts both as a maturase and as a homing endonuclease (I-ScaI) promoting intron mobility. The protein encoded by intron bi2 belongs to a large gene family characterized by the presence of two conserved LAGLIDADG motifs (P1 and P2). In this study, we analysed a set of splicing-deficient mutants of the S. cerevisiae intron bi2 that carry non-directed mutations affecting the maturase activity, and a set of directed missense mutations introduced into the bifunctional protein encoded by the S. capensis intron. Analysis of these mutations has allowed identification of the residues in the conserved P1 and P2 motifs which are crucial for splicing and homing activities. Moreover, several mutations which are located in the C-terminal part of the protein have been found to affect both functions.

Dynamic evolution of plant mitochondrial genomes: Mobile genes and introns and highly variable mutation rates

We summarize our recent studies showing that angiosperm mitochondrial (mt) genomes have experienced remarkably high rates of gene loss and concomitant transfer to the nucleus and of intron acquisition by horizontal transfer. Moreover, we find substantial lineage-specific variation in rates of these structural mutations and also point mutations. These findings mostly arise from a Southern blot survey of gene and intron distribution in 281 diverse angiosperms. These blots reveal numerous losses of mt ribosomal protein genes but, with one exception, only rare loss of respiratory genes. Some lineages of angiosperms have kept all of their mt ribosomal protein genes whereas others have lost most of them. These many losses appear to reflect remarkably high (and variable) rates of functional transfer of mt ribosomal protein genes to the nucleus in angiosperms. The recent transfer of cox2 to the nucleus in legumes provides both an example of interorganellar gene transfer in action and a starting point for discussion of the roles of mechanistic and selective forces in determining the distribution of genetic labor between organellar and nuclear genomes. Plant mt genomes also acquire sequences by horizontal transfer. A striking example of this is a homing group I intron in the mt cox1 gene. This extraordinarily invasive mobile element has probably been acquired over 1,000 times separately during angiosperm evolution via a recent wave of cross-species horizontal transfers. Finally, whereas all previously examined angiosperm mtDNAs have low rates of synonymous substitutions, mtDNAs of two distantly related angiosperms have highly accelerated substitution rates.

Purification and characterization of the DNA cleavage and recognition site of I-ScaI mitochondrial group I intron encoded endonuclease produced in Escherichia coli.

The second intron in the mitochondrial cytb gene of Saccharomyces capensis, belonging to group I, encodes a 280 amino acid protein containing two LAGLIDADG motifs. Genetic and molecular studies have previously shown that this protein has a dual function in the wild-type strain. It acts as a specific homing endonuclease I- Sca I promoting intron mobility and as a maturase promoting intron splicing. Here we describe the synthesis of a universal code equivalent to the mitochondrial sequence coding for this protein and the in vitro characterization of I- Sca I endonuclease activity, using a truncated mutant form of the protein p28bi2 produced in Escherichia coli. We have also determined the cleavage pattern as well as the recognition site of p28bi2. It was found that p28bi2 generates a double-strand cleavage downstream from the intron insertion site with 4 nt long 3'-overhangs. Mutational analysis of the DNA target site shows that p28bi2 recognizes a 16-19 bp sequence from positions -11 to +8 with respect to the intron insertion site.

Recombinant mitochondrial DNA molecules suggest a template switching ability for group-II-intron reverse transcriptase.

Degenerative processes in the filamentous fungus Podospora anserina are strongly correlated with the instability of the mitochondrial genome. Among the sources of instability is the mobile group-II intron COX1-i1, also called intron alpha, which encodes a protein with a reverse transcriptase activity. In this paper we characterize, through PCR experiments, mitochondrial recombinant DNA molecules joining the 5' end of intron alpha to the 3' end of tRNA sequences including the CCA motif. The structure of these junctions led us to propose that they were most probably initiated by a RNA template switching of the reverse transcriptase encoded in COX1-i1. This activity might be involved in a number of mitochondrial rearrangements occurring in degenerative syndromes and in some long-lived mutants.

A Reverse Transcriptase/Maturase Promotes Splicing by Binding at Its Own Coding Segment in a Group II Intron RNA

Group II introns encode reverse transcriptases that promote RNA splicing (maturase activity) and then with the excised intron form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription (TPRT). Here, we show that the primary binding site for the maturase (LtrA) encoded by the Lactococcus lactis Ll.LtrB intron is within a region of intron domain IV that includes the start codon of the LtrA ORF. This binding is enhanced by other elements, particularly domain I and the EBS/IBS interactions, and helps position LtrA to initiate cDNA synthesis in the 3′ exon as occurs during TPRT. Our results suggest how the maturase functions in RNA splicing and support the hypothesis that the reverse transcriptase coding region was derived from an independent genetic element that was inserted into a preexisting group II intron.

A novel group-II intron in the cox1 gene of the fission yeast Schizosaccharomyces pombe is inserted in the same codon as the mobile group-II intron aI2 in the Saccharomyces cerevisiae cox1 homologue

We describe herein a large group-II intron which is inserted in the mitochondrial cox1 gene of the Schizosaccharomyces pombe strain EF2. The intron RNA consists of 2492 nucleotides which can be folded into a secondary structure with all the expected sequence motifs of subgroup-IIA1 introns (Michel et al. 1989). Determination of the exact splice point revealed that the intron is inserted in the same codon, but 1 bp downstream, as the mobile intron aI2 in the Saccharomyces cerevisiae cox1 homologue. A total of nine nucleotide changes was observed around the insertion site of the intron in the cox1 gene of strain EF2 compared with the reference strain ade7-50h(-). Seven of these changes are clustered within the 51 bp upstream of the splice point. Only one sequence deviation was found in the downstream exon. The intron is capable of splicing despite the fact that both the EBS1/IBS1 and the EBS2/IBS2 sequence motifs, thought to be necessary for correct splicing, extend over 5 instead of 6 bp. The maturase, endonuclease and reverse transcriptase domains of the putative protein encoded by the newly described S. pombe group-II intron were not closer to those encoded by the other two, cobI and cox2I, S. pombe group-II introns than to the group-II intron-encoded proteins in Allomyces, Marchantia, Podospora and Saccharomyces.

RNA and protein catalysis in group II intron splicing and mobility reactions using purified components.

Group II introns encode proteins with reverse transcriptase activity. These proteins also promote RNA splicing (maturase activity) and then, with the excised intron, form a site-specific DNA endonuclease that promotes intron mobility by reverse splicing into DNA followed by target DNA-primed reverse transcription. Here, we used an Escherichia coli expression system for the Lactococcus lactis group II intron Ll.LtrB to show that the intron-encoded protein (LtrA) alone is sufficient for maturase activity, and that RNP particles containing only the LtrA protein and excised intron RNA have site-specific DNA endonuclease and target DNA-primed reverse transcriptase activity. Detailed analysis of the splicing reaction indicates that LtrA is an intron-specific splicing factor that binds to unspliced precursor RNA with a K(d) of </=0.12 pM at 30 degrees C. This binding occurs in a rapid bimolecular reaction, which is followed by a slower step, presumably an RNA conformational change, required for splicing to occur. Our results constitute the first biochemical analysis of protein-dependent splicing of a group II intron and demonstrate that a single intron-encoded protein can interact with the intron RNA to carry out a coordinated series of reactions leading to splicing and mobility.

Group II intron reverse transcriptase in yeast mitochondria. Stabilization and regulation of reverse transcriptase activity by the intron RNA

Group II introns encode reverse transcriptases that function in both intron mobility and RNA splicing. The proteins bind specifically to unspliced precursor RNA to promote splicing, and then remain associated with the excised intron to form a DNA endonuclease that mediates intron mobility by target DNA-primed reverse transcription. Here, immunoblotting and UV cross-linking experiments show that the reverse transcriptase activity encoded by the yeast mtDNA group II intron aI2 is associated with an intron-encoded protein of 62 kDa (p62). p62 is bound tightly to endogenous RNAs in mitochondrial ribonucleoprotein particles, and the reverse transcriptase activity is rapidly and irreversibly lost when the protein is released from the endogenous RNAs by RNase digestion. Non-denaturing gel electrophoresis and activity assays show that the aI2 reverse transcriptase is associated predominantly with the excised intron RNA, while a smaller amount is associated with unspliced precursor RNA, as expected from the role of the protein in RNA splicing. Although the reverse transcriptase in wild-type yeast strains is bound tightly to endogenous RNAs, it is regulated so that it does not copy these RNAs unless a suitable DNA oligonucleotide primer or DNA target site is provided. Certain mutations in the intron-encoded protein or RNA circumvent this regulation and activate reverse transcription of endogenous RNAs in the absence of added primer. Although p62 is bound to unspliced precursor RNA in position to initiate cDNA synthesis in the 3′ exon, the major template for target DNA-primed reverse transcription in vitro is the reverse-spliced intron RNA, as found previously for aI1. Together, our results show that binding to intron-containing RNAs stabilizes and regulates the activity of p62.