Toward predicting self-splicing and protein-facilitated splicing of group I introns

In the current era of massive discoveries of noncoding RNAs within genomes, being able to infer a function from a nucleotide sequence is of paramount interest. Although studies of individual group I introns have identified self-splicing and nonself-splicing examples, there is no overall understanding of the prevalence of self-splicing or the factors that determine it among the >2300 group I introns sequenced to date. Here, the self-splicing activities of 12 group I introns from various organisms were assayed under six reaction conditions that had been shown previously to promote RNA catalysis for different RNAs. Besides revealing that assessing self-splicing under only one condition can be misleading, this survey emphasizes that in vitro self-splicing efficiency is correlated with the GC content of the intron (>35% GC was generally conductive to self-splicing), and with the ability of the introns to form particular tertiary interactions. Addition of the Neurospora crassa CYT-18 protein activated splicing of two nonself-splicing introns, but inhibited the second step of self-splicing for two others. Together, correlations between sequence, predicted structure and splicing begin to establish rules that should facilitate our ability to predict the self-splicing activity of any group I intron from its sequence.     

Minimal catalytic domain of a group I self-splicing intron RNA

The self-splicing intron ribozymes have been regarded as primitive forms of the splicing machinery for eukaryotic pre-mRNAs. The splicing activity of group I self-splicing introns is dependent on an absolutely conserved and exceptionally densely packed core region composed of two helical domains, P3-P7 and P4-P6, that are connected rigidly via base triples. Here we show that a mutant group I intron ribozyme lacking both the P4-P6 domain and the base triples can perform the phosphoester transfer reactions required for splicing at both the 5' and 3' splice sites, demonstrating that the elements required for splicing are concentrated in the stacked helical P3-P7 domain. This finding establishes that the conserved core of the intron consists of two physically and functionally separable components, and we present a model showing the architecture of a prototype of this class of intron and the course of its molecular evolution.     

Inhibition of self-splicing group I intron RNA: high-throughput screening assays.

High-throughput screening assays have been developed to rapidly identify small molecule inhibitors targeting catalytic group I introns. Biochemical reactions catalyzed by a self-splicing group I intron derived from Pneumocystis carinii or from bacteriophage T4 have been investigated. In vitro biochemical assays amenable to high-throughput screening have been established. Small molecules that inhibit the functions of group I introns have been identified. These inhibitors should be useful in better understanding ribozyme catalysis or in therapeutic intervention of group I intron-containing microorganisms.     

Requirements for self-splicing of a group I intron from Physarum polycephalum.

The third intron from Physarum polycephalum (Pp LSU 3) is one of the closest known relatives to the well-studied Tetrahymena group I intron. Both introns are located at the same position in the 26S rRNA gene, and with the exception of an open reading frame in Pp LSU 3, are highly homologous. While Pp LSU 3 has been shown to self splice, little is known about its activity in vitro. We have examined the requirements for self splicing in greater detail. Despite its similarity to the Tetrahymena intron, Pp LSU 3 is 1500-fold less reactive, demonstrates a preference for high salt, and exhibits a low Km for GTP. Removal of the open reading frame results in a modest increase of activity. This system provides an opportunity to understand how sequence variations in two related introns alter the efficiency of autoexcision, and how this relates to adaptation of group I introns to their particular sequence context.     

An allosteric-feedback mechanism for protein-assisted group I intron splicing

The I-AniI maturase facilitates self-splicing of a mitochondrial group I intron in Aspergillus nidulans. Binding occurs in at least two steps: first, a specific but labile encounter complex rapidly forms and then this intermediate is slowly resolved into a native, catalytically active RNA/protein complex. Here we probe the structure of the RNA throughout the assembly pathway. Although inherently unstable, the intron core, when bound by I-AniI, undergoes rapid folding to a near-native state in the encounter complex. The next transition includes the slow destabilization and docking into the core of the peripheral stacked helix that contains the 5' splice site. Mutational analyses confirm that both transitions are important for native complex formation. We propose that protein-driven destabilization and docking of the peripheral stacked helix lead to subtle changes in the I-AniI binding site that facilitate native complex formation. These results support an allosteric-feedback mechanism of RNA-protein recognition in which proteins engaged in an intermediate complex can influence RNA structure far from their binding sites. The linkage of these changes to stable binding ensures that the protein and RNA do not get sequestered in nonfunctional complexes.     

A novel mechanism for protein-assisted group I intron splicing

Previously it was shown that the Aspergillus nidulans (A.n.) mitochondrial COB intron maturase, I-AniI, facilitates splicing of the COB intron in vitro. In this study, we apply kinetic analysis of binding and splicing along with RNA deletion analysis to gain insight into the mechanism of I-AniI facilitated splicing. Our results are consistent with I-AniI and A.n. COB pre-RNA forming a specific but labile encounter complex that is resolved into the native, splicing-competent complex. Significantly, kinetic analysis of splicing shows that the resolution step is rate limiting for splicing. RNA deletion studies show that I-AniI requires most of the A.n. COB intron for binding suggesting that the integrity of the I-AniI-binding site depends on overall RNA tertiary structure. These results, taken together with the observation that A.n. COB intron lacks significant stable tertiary structure in the absence of protein, support a model in which I-AniI preassociates with an unfolded COB intron via a "labile" interaction that facilitates correct folding of the intron catalytic core, perhaps by resolving misfolded RNAs or narrowing the number of conformations sampled by the intron during its search for native structure. The active intron conformation is then "locked in" by specific binding of I-Anil to its intron interaction site.     

Minimum secondary structure requirements for catalytic activity of a self-splicing group I intron

We have completed a comprehensive deletion analysis of the Tetrahymena ribozyme in order to define the minimum secondary structure requirements for phosphoester transfer activity of a self-splicing group I intron. A total of 299 nucleotides were removed in a piecewise fashion, leaving a catalytic core of 114 nucleotides that form 7 base-paired structural elements. Among the various deletion mutants are a 300-nucleotide single-deletion mutant and a 281-nucleotide double-deletion mutant whose activity exceeds that of the wild type when tested under physiologic conditions. Consideration of those structural elements that are essential for catalytic activity leads to a simplified secondary structure model of the catalytic core of a group I intron.     

A peripheral element assembles the compact core structure essential for group I intron self-splicing

The presence of non-conserved peripheral elements in all naturally occurring group I introns underline their importance in ensuring the natural intron function. Recently, we reported that some peripheral elements are conserved in group I introns of IE subgroup. Using self-splicing activity as a readout, our initial screening revealed that one such conserved peripheral elements, P2.1, is mainly required to fold the catalytically active structure of the Candida ribozyme, an IE intron. Unexpectedly, the essential function of P2.1 resides in a sequence-conserved short stem of P2.1 but not in a long-range interaction associated with the loop of P2.1 that stabilizes the ribozyme structure. The P2.1 stem is indispensable in folding the compact ribozyme core, most probably by forming a triple helical interaction with two core helices, P3 and P6. Surprisingly, although the ribozyme lacking the P2.1 stem renders a loosely folded core and the loss of self-splicing activity requires two consecutive transesterifications, the mutant ribozyme efficiently catalyzes the first transesterification reaction. These results suggest that the intron self-splicing demands much more ordered structure than does one independent transesterification, highlighting that the universally present peripheral elements achieve their functional importance by enabling the highly ordered structure through diverse tertiary interactions.     

Mutational analysis of conserved nucleotides in a self-splicing group I intron.

We have constructed all single base substitutions in almost all of the highly conserved residues of the Tetrahymena self-splicing intron. Mutation of highly conserved residues almost invariably leads to loss of enzymatic activity. In many cases, activity could be regained by making additional mutations that restored predicted base-pairings; these second site suppressors in general confirm the secondary structure derived from phylogenetic data. At several positions, our suppression data can be most readily explained by assuming non-Watson-Crick base-pairings. In addition to the requirements imposed by the secondary structure, the sequence of the intron is constrained by "negative interactions", the exclusion of particular nucleotide sequences that would form undesirable secondary structures. A comparison of genetic and phylogenetic data suggests sites that may be involved in tertiary structural interactions.     

New approaches to targeting RNA with oligonucleotides: inhibition of group I intron self-splicing

RNA is one class of relatively unexplored drug targets. Since RNAs play a myriad of essential roles, it is likely that new drugs can be developed that target RNA. There are several factors that make targeting RNA particularly attractive. First, the amount of information about the roles of RNA in essential biological processes is currently being expanded. Second, sequence information about targetable RNA is pouring out of genome sequencing efforts at unprecedented levels. Third, designing and screening potential oligonucleotide therapeutics to target RNA is relatively simple. The use of oligonucleotides in cell culture, however, presents several challenges such as oligonucleotide uptake and stability, and selective targeting of genes of interest. Here, we review investigations aimed at targeting RNA with oligonucleotides that can circumvent several of these potential problems. The hallmark of the strategies discussed is the use of short oligonucleotides, which may have the advantage of higher cellular uptake and improved binding selectivity compared to longer oligonucleotides. These strategies have been applied to Group I introns from the mammalian pathogens Pneumocystis carinii and Candida albicans. Both are examples of fungal infections that are increasing in number and prevalence.     

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.     

Crystal structure of a group I intron splicing intermediate

A recently reported crystal structure of an intact bacterial group I self-splicing intron in complex with both its exons provided the first molecular view into the mechanism of RNA splicing. This intron structure, which was trapped in the state prior to the exon ligation reaction, also reveals the architecture of a complex RNA fold. The majority of the intron is contained within three internally stacked, but sequence discontinuous, helical domains. Here the tertiary hydrogen bonding and stacking interactions between the domains, and the single-stranded joiner segments that bridge between them, are fully described. Features of the structure include: (1) A pseudoknot belt that circumscribes the molecule at its longitudinal midpoint; (2) two tetraloop-tetraloop receptor motifs at the peripheral edges of the structure; (3) an extensive minor groove triplex between the paired and joiner segments, P6-J6/6a and P3-J3/4, which provides the major interaction interface between the intron's two primary domains (P4-P6 and P3-P9.0); (4) a six-nucleotide J8/7 single stranded element that adopts a mu-shaped structure and twists through the active site, making critical contacts to all three helical domains; and (5) an extensive base stacking architecture that realizes 90% of all possible stacking interactions. The intron structure was validated by hydroxyl radical footprinting, where strong correlation was observed between experimental and predicted solvent accessibility. Models of the pre-first and pre-second steps of intron splicing are proposed with full-sized tRNA exons. They suggest that the tRNA undergoes substantial angular motion relative to the intron between the two steps of splicing.     

A self-splicing group I intron in DNA polymerase genes of T7-like bacteriophages

Group I introns are inserted into genes of a wide variety of bacteriophages of gram-positive bacteria. However, among the phages of enteric and other gram-negative proteobacteria, introns have been encountered only in phage T4 and several of its close relatives. Here we report the insertion of a self-splicing group I intron in the coding sequence of the DNA polymerase genes of PhiI and W31, phages that are closely related to T7. The introns belong to subgroup IA2 and both contain an open reading frame, inserted into structural element P6a, encoding a protein belonging to the HNH family of homing endonucleases. The introns splice efficiently in vivo and self-splice in vitro under mild conditions of ionic strength and temperature. We conclude that there is no barrier for maintenance of group I introns in phages of proteobacteria.     

Probing the role of a secondary structure element at the 5'- and 3'-splice sites in group I intron self-splicing: the tetrahymena L-16 ScaI ribozyme reveals a new role of the G.U pair in self-splicing

Several ribozyme constructs have been used to dissect aspects of the group I self-splicing reaction. The Tetrahymena L-21 ScaI ribozyme, the best studied of these intron analogues, catalyzes a reaction analogous to the first step of self-splicing, in which a 5'-splice site analogue (S) and guanosine (G) are converted into a 5'-exon analogue (P) and GA. This ribozyme preserves the active site but lacks a short 5'-terminal segment (called the IGS extension herein) that forms dynamic helices, called the P1 extension and P10 helix. The P1 extension forms at the 5'-splice site in the first step of self-splicing, and P10 forms at the 3'-splice site in the second step of self-splicing. To dissect the contributions from the IGS extension and the helices it forms, we have investigated the effects of each of these elements at each reaction step. These experiments were performed with the L-16 ScaI ribozyme, which retains the IGS extension, and with 5'- and 3'-splice site analogues that differ in their ability to form the helices. The presence of the IGS extension strengthens binding of P by 40-fold, even when no new base pairs are formed. This large effect was especially surprising, as binding of S is essentially unaffected for S analogues that do not form additional base pairs with the IGS extension. Analysis of a U.U pair immediately 3' to the cleavage site suggests that a previously identified deleterious effect from a dangling U residue on the L-21 ScaI ribozyme arises from a fortuitous active site interaction and has implications for RNA tertiary structure specificity. Comparisons of the affinities of 5'-splice site analogues that form only a subset of base pairs reveal that inclusion of the conserved G.U base pair at the cleavage site of group I introns destabilizes the P1 extension >100-fold relative to the stability of a helix with all Watson-Crick base pairs. Previous structural data with model duplexes and the recent intron structures suggest that this effect can be attributed to partial unstacking of the P1 extension at the G.U step. These results suggest a previously unrecognized role of the G.U wobble pair in self-splicing: breaking cooperativity in base pair formation between P1 and the P1 extensions. This effect may facilitate replacement of the P1 extension with P10 after the first chemical step of self-splicing and release of the ligated exons after the second step of self-splicing.     

A self-splicing group I intron in the DNA polymerase gene of Bacillus subtilis bacteriophage SPO1

We report a self-splicing intron in bacteriophage SPO1, whose host is the gram-positive Bacillus subtilis. The intron contains all the conserved features of primary sequence and secondary structure previously described for the group IA introns of eukaryotic organelles and the gram-negative bacteriophage T4. The SPO1 intron contains an open reading frame of 522 nucleotides. As in the T4 introns, this open reading frame begins in a region that is looped out of the secondary structure, but ends in a highly conserved region of the intron core. The exons encode SPO1 DNA polymerase, which is highly similar to E. coli DNA polymerase I. The demonstration of self-splicing introns in viruses of both gram-positive and gram-negative eubacteria lends further evidence for their early origin in evolution.