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PubMed1995

The Euglena gracilis intron-encoded mat2 locus is interrupted by three additional group II introns.

The 4,144 nt Euglena gracilis chloroplast psbC intron 2 has been characterized as a single, cis-spliced 593 nt group II intron interrupted by an open reading frame of 758 codons in the loop region of domain IV. The 2,277 nt coding region of orf 758 is interrupted by two additional group II introns of 369 nt and 352 nt. Another 553 nt group II intron is located in the 5' untranslated leader region of orf 758. Because the psbC intron 2 orf encodes a maturase-like protein that has reverse transcriptase domains and a domain X characteristic of group II intron-encoded proteins, the locus has been designated mat2. The psbC intron 2 is the first member of a new category of twintron, characterized by introns within a gene within another intron. A potential role of psbC intron 2 as a "founder" intron involved in the spread of introns to new sites in the plastid genome of the Euglenophycae is discussed.

Amino Acid Sequence↗

PubMed2005

Housekeeping recA gene interrupted by group II intron in the thermophilic Geobacillus kaustophilus.

Most of group II introns are found in intergenes and CDSs with unknown functions, but not in housekeeping genes. In particular, no group II intron within the housekeeping recA gene has been reported either in eukaryotic genomes or in prokaryotic genomes. In this study, we found that the recA gene of the thermophilic Geobacillus kaustophilus genome is interrupted by a group II intron (Gk. Int1), and that Gk.Int1 can splice in temperatures above 70 degrees C in vivo. Here, we report the first prokaryotic group II intron to be found in a housekeeping gene, the characteristics of its self-splicing in vivo and in vitro, and our conclusion that the recA gene functions through the self-splicing of Gk.Int1. It is suggested that the amelioration of Gk.Int1 intron has occurred recently, and that it is still in the process of evolution to the recipient genome.

Amino Acid Sequence↗

PubMed2001

Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites.

Group II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements. In addition to "homing" to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site. Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination. Here, by using an "inverted-site" strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site. This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes.

Base Sequence↗

PubMed1997

Extensive structural conservation exists among several homologs of two Euglena chloroplast group II introns.

82 of the 155 chloroplast introns in Euglena gracilis have been categorized as group II introns. Because they are shorter and more divergent than group II introns from other organisms, the assignment of these Euglena introns to the group II class has been questioned. In the current study, two homologs of E. gracilis petB intron 1 and four homologs of psbC intron 2 have been isolated from related species and characterized. Based on a comparative sequence analysis of intron homologs, the intron core and four of the six helical domains present in the canonical group II intron structural model are conserved in E. gracilis petB intron 1 and psbC intron 2 and all of their homologs. Distal portions of domain I, which are involved in most of the tertiary interactions, are less well conserved than the central core.

Animals↗

PubMed1997

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.

Biopolymers↗

PubMed2006

Fluorescence and solution NMR study of the active site of a 160-kDa group II intron ribozyme.

We have reconstructed the group II intron from Pylaiella littoralis (PL) into a hydrolytic ribozyme, comprising domains 1-3 (D123) connected in cis plus domain 5 (D5) supplied in trans that efficiently cleaves spliced exon substrates. Using a novel gel-based fluorescence assay and nuclear magnetic resonance (NMR) spectroscopy, we monitored the direct binding of D5 to D123, characterized the kinetics of the spliced exon hydrolysis reaction (which is mechanistically analogous to the reverse of the second catalytic step of splicing), and identified the binding surface of D123 on D5. This PL ribozyme acts as an RNA endonuclease even at low monovalent (100 mM KCl) and divalent ion concentrations (1-10 mM MgCl(2)). This is in contrast to other group II intron ribozyme systems that require high levels of salt, making NMR analysis problematic. D5 binds tightly to D123 with a K(d) of 650 +/- 250 nM, a K(m) of approximately 300 nM, and a K(cat) of 0.02 min(-1) under single turnover conditions. Within the approximately 160-kDa D123-D5 binary complex, site-specific binding to D123 leads to dramatic chemical shift perturbation of residues localized to the tetraloop and internal bulge within D5, suggesting a structural switch model for D5-assisted splicing. This minimal ribozyme thus recapitulates the essential features of the reverse of the second catalytic step and represents a well-behaved system for ongoing high-resolution structural work to complement folding and catalytic functional studies.

Binding Sites↗

PubMed2006

Excision of the Sinorhizobium meliloti group II intron RmInt1 as circles in vivo.

Excision of group II introns as circles has been described only for a few eukaryotic introns and little is known about the mechanisms involved, the relevance or consequences of the process. We report that splicing of the bacterial group II intron RmInt1 in vivo leads to the formation of both intron lariat and intron RNA circles. We determined that besides being required for the intron splicing reaction, the maturase domain of the intron-encoded protein also controls the balance between lariat and RNA intron circle production. Furthermore, comparison with in vitro self-splicing products indicates that in vivo, the intron-encoded protein appears to promote the use of a correct EBS1/IBS1 intron-exon interaction as well as cleavage at, or next to, the expected 3' splice site. These findings provide new insights on the mechanism of excision of group II introns as circles.

Alternative Splicing↗

PubMed2006

The receptor for branch-site docking within a group II intron active site.

The distinguishing feature of group II introns, and the property that links them with spliceosomal catalysis, is their ability to undergo splicing through branching. In this reaction, the 2'-hydroxyl group of a specific adenosine within intron domain 6 serves as the nucleophile for attack on the 5' splice site. We know less about branching than any other feature of group II intron catalysis, largely because the receptor structure for activating the branch site is unknown. Here, we identify the intronic region that binds the branch site of a group IIB intron. Located in domain 1, close to receptors for intron domain 5 and both splice sites, we demonstrate that the branch-site receptor is a functional element required for transesterification. Furthermore, we show that crosslinked branch sites can carry out both steps of splicing, suggesting that the conformational state of the intron core is set early and that it persists throughout the entire splicing process.

Binding Sites↗

PubMed1998

Group II intron splicing in vivo by first-step hydrolysis.

Group I, group II and spliceosomal introns splice by two sequential transesterification reactions. For both spliceosomal and group II introns, the first-step reaction occurs by nucleophilic attack on the 5' splice junction by the 2' hydroxyl of an internal adenosine, forming a 2'-5' phosphodiester branch in the intron. The second reaction joins the two exons with a 3'-5' phosphodiester bond and releases intron lariat. In vitro, group II introns can self-splice by an efficient alternative pathway in which the first-step reaction occurs by hydrolysis. The resulting linear splicing intermediate participates in normal second-step reactions, forming spliced exon and linear intron RNAs. Here we show that the group II intron first-step hydrolysis reaction occurs in vivo in place of transesterification in the mitochondria of yeast strains containing branch-site mutations. As expected, the mutations block branching, but surprisingly still allow accurate splicing. This hydrolysis pathway may have been a step in the evolution of splicing mechanisms.

Esterification↗

PubMed1999

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.

Bacterial Proteins↗

PubMed2005

Analysis of the genome of Azotobacter vinelandii revealed the presence of two genetically distinct group II introns on the chromosome.

Azotobacter vinelandii belongs to the y subdivision of eubacteria and has one of the highest respiratory rates. It is considered to be among the probable progenitors of mitochondria. Group II introns were originally identified on organelle genomes. Analysis of the A. vinelandii genome for the presence of group II introns using a deduced group II intron consensus sequence identified two putative introns. The first intron (AVI) which was found to be inserted in the groEL, an essential gene, was already characterized. Our study identified another group II intron (AV2) in A. vinelandii genome. This intron is inserted in a mobile genetic element, similar to most of the group II introns in bacteria, which in this case is a transposase like gene, tnpAl. This putative TnpAl protein is 52% identical to TnpA, the transposase of bacteriophage Lambda, and 85% identical to TnpAl of Pseudomonas stutzeri. Sequence analysis showed that this intron encodes a reverse transcriptase (RT) like motif in domain IV, similar to other group II introns. The RT of this intron open reading frame (ORF) is 53% homologous with that of AVI intron and 66% homologous with that of Pseudomonas putida (Tn5041c) intron. Secondary structure analysis showed that this intron has the typical sub-group IIB1 structure, but the EBS2-IBS2 interaction appears to be missing. Using the RNA generated by in vitro transcription of the intron sequence with its flanking exons, in vitro splicing experiments were performed. It was found that the AV2 intron is functional, despite of lacking the EBS2-IBS2 interaction that plays a role in exon recognition.

Amino Acid Sequence↗

PubMed2005

Unusual group II introns in bacteria of the Bacillus cereus group.

A combination of sequence and structure analysis and reverse transcriptase PCR experiments was used to characterize the group II introns in the complete genomes of two strains of the pathogen Bacillus cereus. While B. cereus ATCC 14579 harbors a single intron element in the chromosome, B. cereus ATCC 10987 contains three introns in the chromosome and four in its 208-kb pBc10987 plasmid. The most striking finding is the presence in B. cereus ATCC 10987 of an intron [B.c.I2(a)] located on the reverse strand of a gene encoding a putative cell surface protein which appears to be correlated to strains of clinical origin. Because of the opposite orientation of B.c.I2(a), the gene is disrupted. Even more striking is that B.c.I2(a) splices out of an RNA transcript corresponding to the opposite DNA strand. All other intragenic introns studied here are inserted in the same orientation as their host genes and splice out of the mRNA in vivo, setting the flanking exons in frame. Noticeably, B.c.I3 in B. cereus ATCC 10987 represents the first example of a group II intron entirely included within a conserved replication gene, namely, the alpha subunit of DNA polymerase III. Another striking finding is that the observed 3' splice site of B.c.I4 occurs 56 bp after the predicted end of the intron. This apparently unusual splicing mechanism may be related to structural irregularities in the 3' terminus. Finally, we also show that the intergenic introns of B. cereus ATCC 10987 are transcribed with their upstream genes and do splice in vivo.

Bacillus cereus↗

PubMed2006

Arabidopsis orthologs of maize chloroplast splicing factors promote splicing of orthologous and species-specific group II introns.

Chloroplast genomes in plants and green algae contain numerous group II introns, large ribozymes that splice via the same chemical steps as spliceosome-mediated splicing in the nucleus. Most chloroplast group II introns are degenerate, requiring interaction with nucleus-encoded proteins to splice in vivo. Genetic approaches in maize (Zea mays) and Chlamydomonas reinhardtii have elucidated distinct sets of proteins that assemble with chloroplast group II introns and facilitate splicing. Little information is available, however, concerning these processes in Arabidopsis (Arabidopsis thaliana). To determine whether the paucity of data concerning chloroplast splicing factors in Arabidopsis reflects a fundamental difference between protein-facilitated group II splicing in monocot and dicot plants, we examined the mutant phenotypes associated with T-DNA insertions in Arabidopsis genes encoding orthologs of the maize chloroplast splicing factors CRS1, CAF1, and CAF2 (AtCRS1, AtCAF1, and AtCAF2). We show that the splicing functions and intron specificities of these proteins are largely conserved between maize and Arabidopsis, indicating that these proteins were recruited to promote the splicing of plastid group II introns prior to the divergence of monocot and dicot plants. We show further that AtCAF1 promotes the splicing of two group II introns, rpoC1 and clpP-intron 1, that are found in Arabidopsis but not in maize; AtCAF1 is the first splicing factor described for these introns. Finally, we show that a strong AtCAF2 allele conditions an embryo-lethal phenotype, adding to the body of data suggesting that cell viability is more sensitive to the loss of plastid translation in Arabidopsis than in maize.

Arabidopsis↗

PubMed2004

Molecular evolution and phylogenetic utility of the petD group II intron: a case study in basal angiosperms.

Sequences of spacers and group I introns in plant chloroplast genomes have recently been shown to be very effective in phylogenetic reconstruction at higher taxonomic levels and not only for inferring relationships among species. Group II introns, being more frequent in those genomes than group I introns, may be further promising markers. Because group II introns are structurally constrained, we assumed that sequences of a group II intron should be alignable across seed plants. We designed universal amplification primers for the petD intron and sequenced this intron in a representative selection of 47 angiosperms and three gymnosperms. Our sampling of taxa is the most representative of major seed plant lineages to date for group II introns. Through differential analysis of structural partitions, we studied patterns of molecular evolution and their contribution to phylogenetic signal. Nonpairing stretches (loops, bulges, and interhelical nucleotides) were considerably more variable in both substitutions and indels than in helical elements. Differences among the domains are basically a function of their structural composition. After the exclusion of four mutational hotspots accounting for less than 18% of sequence length, which are located in loops of domains I and IV, all sequences could be aligned unambiguously across seed plants. Microstructural changes predominantly occurred in loop regions and are mostly simple sequence repeats. An indel matrix comprising 241 characters revealed microstructural changes to be of lower homoplasy than are substitutions. In showing Amborella first branching and providing support for a magnoliid clade through a synapomorphic indel, the petD data set proved effective in testing between alternative hypotheses on the basal nodes of the angiosperm tree. Within angiosperms, group II introns offer phylogenetic signal that is intermediate in information content between that of spacers and group I introns on the one hand and coding sequences on the other.

Base Sequence↗

PubMed2003

Group II intron splicing factors derived by diversification of an ancient RNA-binding domain.

Group II introns are ribozymes whose catalytic mechanism closely resembles that of the spliceosome. Many group II introns have lost the ability to splice autonomously as the result of an evolutionary process in which the loss of self-splicing activity was compensated by the recruitment of host-encoded protein cofactors. Genetic screens previously identified CRS1 and CRS2 as host-encoded proteins required for the splicing of group II introns in maize chloroplasts. Here, we describe two additional host-encoded group II intron splicing factors, CRS2-associated factors 1 and 2 (CAF1 and CAF2). We show that CRS2 functions in the context of intron ribonucleoprotein particles that include either CAF1 or CAF2, and that CRS2-CAF1 and CRS2-CAF2 complexes have distinct intron specificities. CAF1, CAF2 and the previously described group II intron splicing factor CRS1 are characterized by similar repeated domains, which we name here the CRM (chloroplast RNA splicing and ribosome maturation) domains. We propose that the CRM domain is an ancient RNA-binding module that has diversified to mediate specific interactions with various highly structured RNAs.

Amino Acid Sequence↗

PubMed2001

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.

Adenine↗

PubMed1995

A UV-induced, Mg(2+)-dependent crosslink traps an active form of domain 3 of a self-splicing group II intron.

In vitro irradiation of a15 gamma group II intron RNA with low doses of 254 nm UV light induces a single major crosslink. This crosslink was mapped within the domain 3 substructure of this RNA and one of the participating nucleotides was identified. When an RNA containing only the domain 3 substructure is irradiated under the same conditions, an intramolecular crosslink forms between two specific pyrimidines, one of them identical to the nucleotide crosslinked in the full-length intron RNA. In both RNAs, the crosslink is magnesium ion-dependent and photoreversible. A trans assay for domain 3 function was developed and used to find that the crosslinked domain 3 RNA remains highly reactive. This suggests that crosslinking has trapped a functional, Mg(2+)-induced folded state of this group II intron substructure and that this folding is probably independent of the other domains of the intron.

Base Sequence↗

PubMed2002

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

BACKGROUND: Group 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. RESULTS: The 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 (DeltaORF 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. CONCLUSIONS: The 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.

Journal Article↗