The tertiary structure of group II introns: implications for biological function and evolution

Group II introns are some of the largest ribozymes in nature, and they are a major source of information about RNA assembly and tertiary structural organization. These introns are of biological significance because they are self-splicing mobile elements that have migrated into diverse genomes and played a major role in the genomic organization and metabolism of most life forms. The tertiary structure of group II introns has been the subject of many phylogenetic, genetic, biochemical and biophysical investigations, all of which are consistent with the recent crystal structure of an intact group IIC intron from the alkaliphilic eubacterium Oceanobacillus iheyensis. The crystal structure reveals that catalytic intron domain V is enfolded within the other intronic domains through an elaborate network of diverse tertiary interactions. Within the folded core, DV adopts an activated conformation that readily binds catalytic metal ions and positions them in a manner appropriate for reaction with nucleic acid targets. The tertiary structure of the group II intron reveals new information on motifs for RNA architectural organization, mechanisms of group II intron catalysis, and the evolutionary relationships among RNA processing systems. Guided by the structure and the wealth of previous genetic and biochemical work, it is now possible to deduce the probable location of DVI and the site of additional domains that contribute to the function of the highly derived group IIB and IIA introns.     

Principles of ion recognition in RNA: insights from the group II intron structures

Metal ions promote both RNA folding and catalysis, thus being essential in stabilizing the structure and determining the function of large RNA molecules, including group II introns. The latter are self-splicing metalloribozymes, containing a heteronuclear four-metal-ion center within the active site. In addition to these catalytic ions, group II introns bind many other structural ions, including delocalized ions that bind the RNA diffusively and well-ordered ions that bind the RNA tightly with high occupancy. The latter ions, which can be studied by biophysical methods, have not yet been analyzed systematically. Here, we compare crystal structures of the group IIC intron from Oceanobacillus iheyensis and classify numerous site-bound ions, which are primarily localized in the intron core and near long-range tertiary contacts. Certain ion-binding sites resemble motifs observed in known RNA structures, while others are idiosyncratic to the group II intron. Particularly interesting are (1) ions proximal to the active site, which may participate in splicing together with the catalytic four-metal-ion center, (2) organic ions that bind regions predicted to interact with intron-encoded proteins, and (3) unusual monovalent ions bound to GU wobble pairs, GA mismatches, the S-turn, the tetraloop-receptor, and the T-loop. Our analysis extends the general principles by which ions participate in RNA structural organization and it will aid in the determination and interpretation of future RNA structures.     

Unwinding by Local Strand Separation Is Critical for the Function of DEAD-Box Proteins as RNA Chaperones

The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are broadly acting RNA chaperones that function in mitochondria to stimulate group I and group II intron splicing and to activate mRNA translation. Previous studies showed that the S. cerevisiae cytosolic/nuclear DEAD-box protein Ded1p could stimulate group II intron splicing in vitro. Here, we show that Ded1p complements mitochondrial translation and group I and group II intron splicing defects in mss116Δ strains, stimulates the in vitro splicing of group I and group II introns, and functions indistinguishably from CYT-19 to resolve different nonnative secondary and/or tertiary structures in the Tetrahymena thermophila large subunit rRNA-ΔP5abc group I intron. The Escherichia coli DEAD-box protein SrmB also stimulates group I and group II intron splicing in vitro, while the E. coli DEAD-box protein DbpA and the vaccinia virus DExH-box protein NPH-II gave little, if any, group I or group II intron splicing stimulation in vitro or in vivo. The four DEAD-box proteins that stimulate group I and group II intron splicing unwind RNA duplexes by local strand separation and have little or no specificity, as judged by RNA-binding assays and stimulation of their ATPase activity by diverse RNAs. In contrast, DbpA binds group I and group II intron RNAs nonspecifically, but its ATPase activity is activated specifically by a helical segment of E. coli 23S rRNA, and NPH-II unwinds RNAs by directional translocation. The ability of DEAD-box proteins to stimulate group I and group II intron splicing correlates primarily with their RNA-unwinding activity, which, for the protein preparations used here, was greatest for Mss116p, followed by Ded1p, CYT-19, and SrmB. Furthermore, this correlation holds for all group I and group II intron RNAs tested, implying a fundamentally similar mechanism for both types of introns. Our results support the hypothesis that DEAD-box proteins have an inherent ability to function as RNA chaperones by virtue of their distinctive RNA-unwinding mechanism, which enables refolding of localized RNA regions or structures without globally disrupting RNA structure.     

A structural analysis of the group II intron active site and implications for the spliceosome

Group II introns are self-splicing, mobile genetic elements that have fundamentally influenced the organization of terrestrial genomes. These large ribozymes remain important for gene expression in almost all forms of bacteria and eukaryotes and they are believed to share a common ancestry with the eukaryotic spliceosome that is required for processing all nuclear pre-mRNAs. The three-dimensional structure of a group IIC intron was recently determined by X-ray crystallography, making it possible to visualize the active site and the elaborate network of tertiary interactions that stabilize the molecule. Here we describe the molecular features of the active site in detail and evaluate their correspondence with prior biochemical, genetic, and phylogenetic analyses on group II introns. In addition, we evaluate the structural significance of RNA motifs within the intron core, such as the major-groove triple helix and the domain 5 bulge. Having combined what is known about the group II intron core, we then compare it with known structural features of U6 snRNA in the eukaryotic spliceosome. This analysis leads to a set of predictions for the molecular structure of the spliceosomal active site.     

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. gracilispetB 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. gracilispetB 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.     

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.     

The Mrs1 Splicing Factor Binds the bI3 Group I Intron at Each of Two Tetraloop-Receptor Motifs

Most large ribozymes require protein cofactors in order to function efficiently. The yeast mitochondrial bI3 group I intron requires two proteins for efficient splicing, Mrs1 and the bI3 maturase. Mrs1 has evolved from DNA junction resolvases to function as an RNA cofactor for at least two group I introns; however, the RNA binding site and the mechanism by which Mrs1 facilitates splicing were unknown. Here we use high-throughput RNA structure analysis to show that Mrs1 binds a ubiquitous RNA tertiary structure motif, the GNRA tetraloop-receptor interaction, at two sites in the bI3 RNA. Mrs1 also interacts at similar tetraloop-receptor elements, as well as other structures, in the self-folding Azoarcus group I intron and in the RNase P enzyme. Thus, Mrs1 recognizes general features found in the tetraloop-receptor motif. Identification of the two Mrs1 binding sites now makes it possible to create a model of the complete six-component bI3 ribonucleoprotein. All protein cofactors bind at the periphery of the RNA such that every long-range RNA tertiary interaction is stabilized by protein binding, involving either Mrs1 or the bI3 maturase. This work emphasizes the strong evolutionary pressure to bolster RNA tertiary structure with RNA-binding interactions as seen in the ribosome, spliceosome, and other large RNA machines.     

The Ll.LtrB intron from Lactococcus lactis excises as circles in vivo: insights into the group II intron circularization pathway

Group II introns are large ribozymes that require the assistance of intron-encoded or free-standing maturases to splice from their pre-mRNAs in vivo. They mainly splice through the classical branching pathway, being released as RNA lariats. However, group II introns can also splice through secondary pathways like hydrolysis and circularization leading to the release of linear and circular introns, respectively. Here, we assessed in vivo splicing of various constructs of the Ll.LtrB group II intron from the Gram-positive bacterium Lactococcus lactis. The study of excised intron junctions revealed, in addition to branched intron lariats, the presence of perfect end-to-end intron circles and alternatively circularized introns. Removal of the branch point A residue prevented Ll.LtrB excision through the branching pathway but did not hinder intron circle formation. Complete intron RNA circles were found associated with the intron-encoded protein LtrA forming nevertheless inactive RNPs. Traces of double-stranded head-to-tail intron DNA junctions were also detected in L. lactis RNA and nucleic acid extracts. Some intron circles and alternatively circularized introns harbored variable number of non-encoded nucleotides at their splice junction. The presence of mRNA fragments at the splice junction of some intron RNA circles provides insights into the group II intron circularization pathway in bacteria.     

A group II intron inserted into a bacterial heat-shock operon shows autocatalytic activity and unusual thermostability

Group II intron RNAs fold into catalytically active structures that catalyze their own self-splicing and subsequent transposition into DNA. Because of their remarkable enzymatic properties, it has been of interest to find new group II introns with novel properties. Here we report the cloning, sequencing, and mechanistic characterization of a new group II intron from the bacterium Azotobacter vinelandii (the AV intron). Although it bears the characteristics of the group IIB1 class, the AV intron is unusually G-C rich, and it has unusual insertion sequences and a minimal dependence on the EBS2-IBS2 tertiary interaction. The AV intron is the first bacterial intron that has been found to reside in a housekeeping gene which, in this case, encodes a heat-shock protein (hsp60). Consistent with a potential role in heat-shock regulation, kinetic analysis reveals that AV intron self-splicing is activated only at elevated temperatures. This suggests a novel pathway for the regulation of heat shock in prokaryotes and provides a first example of a thermally tolerant group II intron RNA.     

Origin and evolution of the chloroplast trnK (matK) intron: a model for evolution of group II intron RNA structures

The trnK intron of plants encodes the matK open reading frame (ORF), which has been used extensively as a phylogenetic marker for classification of plants. Here we examined the evolution of the trnK intron itself as a model for group II intron evolution in plants. Representative trnK intron sequences were compiled from species spanning algae to angiosperms, and four introns were newly sequenced. Phylogenetic analyses showed that the matK ORFs belong to the ML (mitochondrial-like) subclass of group II intron ORFs, indicating that they were derived from a mobile group II intron of the class. RNA structures of the introns were folded and analyzed, which revealed progressive RNA structural deviations and degenerations throughout plant evolution. The data support a model in which plant organellar group II introns were derived from bacterial-like introns that had "standard" RNA structures and were competent for self-splicing and mobility and that subsequently the ribozyme structures degenerated to ultimately become dependent upon host-splicing factors. We propose that the patterns of RNA structure evolution seen for the trnK intron will apply to the other group II introns in plants.     

Group II intron–ribosome association protects intron RNA from degradation

The influence of the cellular environment on the structures and properties of catalytic RNAs is not well understood, despite great interest in ribozyme function. Here we report on ribosome association of group II introns, which are ribozymes that are important because of their putative ancestry to spliceosomal introns and retrotransposons, their retromobility via an RNA intermediate, and their application as gene delivery agents. We show that group II intron RNA, in complex with the intron-encoded protein from the native Lactoccocus lactis host, associates strongly with ribosomes in vivo. Ribosomes have little effect on intron ribozyme activities; rather, the association with host ribosomes protects the intron RNA against degradation by RNase E, an enzyme previously shown to be a silencer of retromobility in Escherichia coli. The ribosome interacts strongly with the intron, exerting protective effects in vivo and in vitro, as demonstrated by genetic and biochemical experiments. These results are consistent with the ribosome influencing the integrity of catalytic RNAs in bacteria in the face of degradative nucleases that regulate 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 ai5gamma. 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 ai5gamma 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 ai5gamma 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.     

A single active-site region for a group II intron

Despite the biological importance of self-splicing group II introns, little is known about their structural organization. Synthetic incorporation of site-specific photo-cross-linkers within catalytic domains resulted in functional distance constraints that, when combined with known tertiary interactions, provide a three-dimensional view of the active intron architecture. All functionalities important for both steps of splicing are proximal before the first step, suggestive of a single active-site region for group II intron catalysis.