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PubMed1998

A map of the binding site for catalytic domain 5 in the core of a group II intron ribozyme.

Group II introns are ribozymes with a complex tertiary architecture that is of great interest as a model for RNA folding. Domain 5 (D5) is a highly conserved region of the intron that is considered one of the most critical structures in the catalytic core. Despite its central importance, the means by which D5 interacts with other core elements is unclear. To obtain a map of potential interaction sites, dimethyl sulfate was used to footprint regions of the intron that are involved in D5 binding. These studies were complemented by measurements of D5 binding to a series of truncated intron derivatives. In this way, the minimal region of the intron required for strong D5 association was defined and the sites most likely to represent thermodynamically significant positions of tertiary contact were identified. These studies show that ground-state D5 binding is mediated by tertiary contacts to specific regions of D1, including a tetraloop receptor and an adjacent three-way junction. In contrast, D2 and D3 are not found to stabilize D5 association. These data highlight the significance of D1-D5 interactions and will facilitate the identification of specific tertiary contacts between them.

Base Sequence↗
PubMed2003

ORF-less and reverse-transcriptase-encoding group II introns in archaebacteria, with a pattern of homing into related group II intron ORFs.

Although group II intron retroelements are prevalent in eubacteria, they have not been identified in archaebacteria in the first 10 genomes sequenced. However, the recently sequenced archael genome of Methanosarcina acetivorans contains 21 group II introns, including 7 introns that do not encode reverse transcriptase ORFs. To our knowledge, these are the first retroelements identified in archaebacteria, and the first ORF-less group II introns in bacteria. Furthermore, the insertion pattern of the introns is highly unusual. The introns appear to insert site-specifically into ORFs of other group II introns, forming nested clusters of up to four introns, but there are no flanking exons that could encode a functional protein after the introns have been spliced out.

Amino Acid Sequence↗
PubMed2003

Domains 2 and 3 interact to form critical elements of the group II intron active site.

Group II introns are self-splicing RNA molecules that also behave as mobile genetic elements. The secondary structure of group II intron RNAs is typically described as a series of six domains that project from a central wheel. Most structural and mechanistic analyses of the intron have focused on domains 1 and 5, which contain the residues essential for catalysis, and on domain 6, which contains the branch-point adenosine. Domains 2 and 3 (D2, D3) have been shown to make important contributions to intronic activity; however, information about their function is quite limited. To elucidate the role of D2 and D3 in group II ribozyme catalysis, we built a series of multi-piece ribozyme constructs based on the ai5gamma group II intron. These constructs are designed to shed light on the roles of D2 and D3 in some of the major reactions catalyzed by the intron: 5'-exon cleavage, branching, and substrate hydrolysis. Reactions with these constructs demonstrate that D3 stimulates the chemical rate constant of group II intron reactions, and that it behaves as a form of catalytic effector. However, D3 is unable to associate independently with the ribozyme core. Docking of D3 is mediated by a short duplex that is found at the base of D2. In addition to recruiting D3 into the core, the D2 stem directs the folding of the adjacent j(2/3) linker, which is among the most conserved elements in the group II intron active site. In turn, the D2 stem contributes to 5'-splice site docking and ribozyme conformational change. Nucleotide analog interference mapping suggests an interaction between the D2 stem and D3 that builds on the known theta-theta' interaction and extends it into D3. These results establish that D3 and the base of D2 are key elements of the group II intron core and they suggest a hierarchy for active-site assembly.

Base Sequence↗
PubMed1996

Group II introns: elaborate ribozymes.

Group II introns are found in organelle genomes of plants, fungi and algae as well as in some bacteria. Some group II introns have been shown to self-splice in vitro and thus constitute examples of ribozymes. Their splicing pathway is analogous to the splicing pathway of nuclear pre-mRNA introns. They thus constitute simple models to analyze RNA catalysis of this type of splicing reactions. In this review article, I will summarize our current state of understanding of the ribozyme activity of group II introns and show that their large size correlates with their ability to perform complex tasks. After discussing the similarities found between group II and nuclear pre-mRNA introns, I will briefly evoke how the ribozyme activity of group II introns might be involved in their transposition at the DNA level.

Binding Sites↗
PubMed2002

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.

Base Sequence↗
PubMed2005

Conjugative transfer of the Lactococcus lactis chromosomal sex factor promotes dissemination of the Ll.LtrB group II intron.

The Ll.LtrB group II intron from the low-G+C gram-positive bacterium Lactococcus lactis was the first bacterial group II intron shown to splice and mobilize in vivo. This retroelement interrupts the relaxase gene (ltrB) of three L. lactis conjugative elements: plasmids pRS01 and pAH90 and the chromosomal sex factor. Conjugative transfer of a plasmid harboring a segment of the pRS01 conjugative plasmid including the Ll.LtrB intron allows dissemination of Ll.LtrB among L. lactis strains and lateral transfer of this retroelement from L. lactis to Enterococcus faecalis. Here we report the dissemination of the Ll.LtrB group II intron among L. lactis strains following conjugative transfer of the native chromosomally embedded L. lactis sex factor. We demonstrated that Ll.LtrB dissemination is highly variable and often more efficient from this integrative and conjugative element than from an engineered conjugative plasmid. Cotransfer among L. lactis strains of both Ll.LtrB-containing elements, the conjugative plasmid and the sex factor, was detected and shown to be synergistic. Moreover, following their concurrent transfer, both mobilizable elements supported the spread of their respective copies of the Ll.LtrB intron. Our findings explain the unusually high efficiency of Ll.LtrB mobility observed following conjugation of intron-containing plasmids.

Chromosomes, Bacterial↗
PubMed2001

Excision of group II introns as circles.

Group II introns are usually removed from precursor RNAs as lariats comprised of a circular component and a short 3' tail. We find that group II introns can also be excised as complete circles. Circle formation requires release of the 3' exon of a splicing substrate, apparently by a trans splicing mechanism. After 3' exon release, the terminal uridine of the intron attacks the 5' splice site, releasing the 5' exon and joining the first and last intron residues by a 2'-5' phosphodiester bond. RNA isolated from yeast mitochondria also contains circles, indicating that at least one group II intron (aI2) forms circles in vivo. Furthermore, analysis of RNA and DNA from certain mutant yeast strains shows that circular DNA introns exist and are produced by reverse transcription of RNA, rather than by ectopic homing.

DNA Ligases↗
PubMed2004

CRS1, a chloroplast group II intron splicing factor, promotes intron folding through specific interactions with two intron domains.

Group II introns are ribozymes that catalyze a splicing reaction with the same chemical steps as spliceosome-mediated splicing. Many group II introns have lost the capacity to self-splice while acquiring compensatory interactions with host-derived protein cofactors. Degenerate group II introns are particularly abundant in the organellar genomes of plants, where their requirement for nuclear-encoded splicing factors provides a means for the integration of nuclear and organellar functions. We present a biochemical analysis of the interactions between a nuclear-encoded group II splicing factor and its chloroplast intron target. The maize (Zea mays) protein Chloroplast RNA Splicing 1 (CRS1) is required specifically for the splicing of the group II intron in the chloroplast atpF gene and belongs to a plant-specific protein family defined by a recently recognized RNA binding domain, the CRM domain. We show that CRS1's specificity for the atpF intron in vivo can be explained by CRS1's intrinsic RNA binding properties. CRS1 binds in vitro with high affinity and specificity to atpF intron RNA and does so through the recognition of elements in intron domains I and IV. These binding sites are not conserved in other group II introns, accounting for CRS1's intron specificity. In the absence of CRS1, the atpF intron has little uniform tertiary structure even at elevated [Mg2+]. CRS1 binding reorganizes the RNA, such that intron elements expected to be at the catalytic core become less accessible to solvent. We conclude that CRS1 promotes the folding of its group II intron target through tight and specific interactions with two peripheral intron segments.

Binding Sites↗
PubMed1990

Involvement of aminoacyl-tRNA synthetases and other proteins in group I and group II intron splicing.

Group I and group II introns catalyse their own splicing, but depend on protein factors for efficient splicing in vivo. Some of these proteins, termed maturases, are encoded by the introns themselves and may also function in intron mobility. Other proteins are encoded by host chromosomal genes and include aminoacyl-tRNA synthetases and various proteins that function in protein synthesis. The splicing factors identified thus far appear to be idiosyncratic, even in closely related organisms. We suggest that some of these protein-assisted splicing reactions evolved relatively recently, possibly reflecting the recent dispersal of the introns themselves.

Amino Acyl-tRNA Synthetases↗
PubMed2001

Mechanism of maturase-promoted group II intron splicing.

Mobile group II introns encode reverse transcriptases that also function as intron-specific splicing factors (maturases). We showed previously that the reverse transcriptase/maturase encoded by the Lactococcus lactis Ll.LtrB intron has a high affinity binding site at the beginning of its own coding region in an idiosyncratic structure, DIVa. Here, we identify potential secondary binding sites in conserved regions of the catalytic core and show via chemical modification experiments that binding of the maturase induces the formation of key tertiary interactions required for RNA splicing. The interaction with conserved as well as idiosyncratic regions explains how maturases in some organisms could evolve into general group II intron splicing factors, potentially mirroring a key step in the evolution of spliceosomal introns.

Aldehydes↗
PubMed1998

Mosaic structure of the cox2 gene in the petite negative yeast Schizosaccharomyces pombe: a group II intron is inserted at the same location as the otherwise unrelated group II introns in the mitochondria of higher plants.

In contrast to homologous genes in other fungal mitochondrial genomes, the gene encoding subunit 2 of cytochrome oxidase (cox2) in several Schizosaccharomyces pombe strains contains a large group II intron. Its 2436 nucleotides can be folded into a typical group II intron secondary structure, possessing all the expected sequence motifs for subgroup IIA1 (Michel et al., 1989). This intron is remarkable for the following reasons: (i) Five nucleotide changes were observed compared with the continuous form of the cox2 gene in the reference strain 50 at the 3'-exon sequence, but not in the 5'-exon. (ii) One of these changes occurred at the splice point leading to a serine instead of a threonine residue in the deduced cox2 polypeptide. In all cases, the alterations resulted in the replacement of more frequently used codons by rare ones. (iii) Although the intron is able to undergo splicing, the sequence motifs thought to be necessary for interaction between the 5'-exon and the intron during the splicing process (the EBS1/IBS1 as well as the EBS2/IBS2 pairings) are unusual. (iv) The intron is inserted at the same location in the cox2 gene as the otherwise unrelated intron from higher plants.

Alleles↗
PubMed2001

Recruitment of a peptidyl-tRNA hydrolase as a facilitator of group II intron splicing in chloroplasts.

Group II introns are catalytic RNAs that have been proposed to be the evolutionary precursors to the spliceosome. Most group II introns require accessory factors to splice efficiently in vivo, but few such factors have been identified. We have cloned the maize nuclear gene crs2, which is required for the splicing of nine group II introns in chloroplasts. CRS2 is related to peptidyl-tRNA hydrolase enzymes. However, CRS2 expression failed to rescue an Escherichia coli pth(ts) mutant and CRS2 lacks several conserved amino acids that are important for the activity of the E.coli enzyme, indicating that it may lack peptidyl-tRNA hydrolase activity. CRS2 is localized to the chloroplast stroma, where it is found in a large salt-stable complex that contains RNA. CRS2 co-sediments with group II intron RNA during centrifugation of stroma through sucrose gradients, suggesting that CRS2 facilitates splicing via direct interaction with intron RNA. Sequence comparisons indicate how evolutionary tinkering may have allowed an enzyme that interacts with peptidyl-tRNAs to acquire a function in group II intron splicing.

Amino Acid Sequence↗
PubMed2000

Group II introns in the bacterial world.

Group II introns are large catalytic RNA molecules that act as mobile genetic elements. They were initially identified in the organelle genomes of lower eukaryotes and plants, and it has been suggested that they are the progenitors of nuclear spliceosomal introns. Group II self-splicing introns were shown to be present in bacteria in 1993, since when the various bacterial genome sequencing projects have led to a significant increase in the number of group II intron sequences present in databases. However, few of these introns have been characterized, and most were identified on the basis of their intron-encoded protein (IEP), with little data available concerning their ribozyme/RNA structure. Their frequency in prokaryotes is also unknown. We attempt here to provide a first comprehensive review of bacterial group II introns based on recent genome sequencing data and mechanistic studies.

Bacteria↗
PubMed2004

GAPDH enhances group II intron splicing in vitro.

Group II introns are autocatalytic RNAs which self-splice in vitro. However, in vivo additional protein factors might be involved in the splicing process. We used an affinity chromatography method called 'StreptoTag' to identify group II intron binding proteins from Saccharomyces cerevisiae. This method uses a hybrid RNA consisting of a streptomycin-binding affinity tag and the RNA of interest, which is bound to a streptomycin column and incubated with yeast protein extract. After several washing steps the bound RNPs are eluted by addition of streptomycin. The eluted RNPs are separated and the proteins identified by mass-spectrometric analysis. Using crude extract from yeast in combination with a substructure of the bl1 group II intron (domains IV-VI) we were able to identify four glycolytic enzymes; glucose-6-phosphate isomerase (GPI), 3-phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and triosephosphate isomerase (TPI). From these proteins GAPDH increases in vitro splicing of the bl1 group II intron by up to three times. However, in vivo GAPDH is not a group II intron-splicing factor, since it is not localised in yeast mitochondria. Therefore, the observed activity reflects an unexpected property of GAPDH. Band shift experiments and UV cross linking demonstrated the interaction of GAPDH with the group II intron RNA. This novel activity expands the reaction repertoire of GAPDH to a new RNA species.

Base Sequence↗
PubMed1990

Reverse self-splicing of group II intron RNAs in vitro.

Group II introns, which are classed together on the basis of a conserved secondary structure, are found in organellar genes of lower eukaryotes and plants. Like introns in nuclear pre-messenger RNA, they are excised by a two-step splicing reaction to generate branched circular RNAs, the so-called lariats. A remarkable feature of group II introns is their self-splicing activity in vitro. In the absence of a nucleotide cofactor, the intron RNAs catalyse two successive transesterification reactions which lead to autocatalytic excision of the lariat IVS from pre-mRNA and concomitantly to exon ligation. By virtue of its ability to specifically bind the 5' exon, the intron can also catalyse such reactions on exogenous RNA substrates. This sequence-specific attachment could enable group II introns to integrate into unrelated RNAs by reverse splicing, in a process similar to that described for the self-splicing Tetrahymena group I intron. Here we report that group II lariat IVS can indeed reintegrate itself into an RNA composed of the ligated exons in vitro. This occurs by a process of self-splicing that completely reverses both transesterification steps of the forward reaction: it involves a transition of the 2'-5' phosphodiester bond of the lariat RNA into the 3'-5' bond of the reconstituted 5' splice junction.

Apoproteins↗
PubMed2000

Retrotransposition of a bacterial group II intron.

Self-splicing group II introns may be the evolutionary progenitors of eukaryotic spliceosomal introns, but the route by which they invade new chromosomal sites is unknown. To address the mechanism by which group II introns are disseminated, we have studied the bacterial L1.LtrB intron from Lactococcus lactis. The protein product of this intron, LtrA, possesses maturase, reverse transcriptase and endonuclease enzymatic activities. Together with the intron, LtrA forms a ribonucleoprotein (RNP) complex which mediates a process known as retrohoming. In retrohoming, the intron reverse splices into a cognate intronless DNA site. Integration of a DNA copy of the intron is recombinase independent but requires all three activities of LtrA. Here we report the first experimental demonstration of a group II intron invading ectopic chromosomal sites, which occurs by a distinct retrotransposition mechanism. This retrotransposition process is endonuclease-independent and recombinase-dependent, and is likely to involve reverse splicing of the intron RNA into cellular RNA targets. These retrotranspositions suggest a mechanism by which splicesomal introns may have become widely dispersed.

Bacterial Proteins↗