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.     

Pentamidine inhibits mitochondrial intron splicing and translation in Saccharomyces cerevisiae

Pentamidine inhibits in vitro splicing of nuclear group I introns from rRNA genes of some pathogenic fungi and is known to inhibit mitochondrial function in yeast. Here we report that pentamidine inhibits the self-splicing of three group I and two group II introns of yeast mitochondria. Comparison of yeast strains with different configurations of mitochondrial introns (12, 5, 4, or 0 introns) revealed that strains with the most introns were the most sensitive to growth inhibition by pentamidine on glycerol medium. Analysis of blots of RNA from yeast strains grown in raffinose medium in the presence or absence of pentamidine revealed that the splicing of seven group I and two group II introns that have intron reading frames was inhibited by the drug to varying extents. Three introns without reading frames were unaffected by the drug in vivo, and two of these were inhibited in vitro, implying that the drug affects splicing by acting directly on RNA in vitro, but on another target in vivo. Because the most sensitive introns in vivo are the ones whose splicing depends on a maturase encoded by the intron reading frames, we tested pentamidine for effects on mitochondrial translation. We found that the drug inhibits mitochondrial but not cytoplasmic translation in cells at concentrations that inhibit mitochondrial intron splicing. Therefore, pentamidine is a potent and specific inhibitor of mitochondrial translation, and this effect explains most or all of its effects on respiratory growth and on in vivo splicing of mitochondrial introns.     

Streptomycin inhibits splicing of group I introns by competition with the guanosine substrate.

Streptomycin is an aminocyclitol glycoside antibiotic, which interferes with prokaryotic protein synthesis by interacting with the ribosomal RNA. We report here that streptomycin is also able to inhibit self splicing of the group I intron of the thymidylate synthase gene of phage T4. The inhibition is kinetically competitive with the substrate guanosine. Streptomycin and guanosine have in common a guanidino group, which has been shown to undergo hydrogen bonds with the ribozyme (Bass & Cech, Biochemistry, 25, 1986, 4473). The inhibitory effect of streptomycin extends to other group I introns, but does not affect group II introns. Mutating the bulged nucleotide in the conserved P7 secondary structure element of the td intron alters the affinity of the ribozyme for both guanosine and streptomycin. Myomycin, an antibiotic with similar effects on protein synthesis as streptomycin, is also able to inhibit splicing. In contrast, bluensomycin, which is structurally related to streptomycin, but contains only one guanidino group does not inhibit splicing. We discuss these findings in support of an evolutionary model that stresses the antiquity of antibiotics (J. Davies, Molecular Microbiology 4, 1990, 1227).     

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.     

The natural history of group I introns

There are four major classes of introns: self-splicing group I and group II introns, tRNA and/or archaeal introns and spliceosomal introns in nuclear pre-mRNA. Group I introns are widely distributed in protists, bacteria and bacteriophages. Group II introns are found in fungal and land plant mitochondria, algal plastids, bacteria and Archaea. Group II and spliceosomal introns share a common splicing pathway and might be related to each other. The tRNA and/or archaeal introns are found in the nuclear tRNA of eukaryotes and in archaeal tRNA, rRNA and mRNA. The mechanisms underlying the self-splicing and mobility of a few model group I introns are well understood. By contrast, the role of these highly distinct processes in the evolution of the 1500 group I introns found thus far in nature (e.g. in algae and fungi) has only recently been clarified. The explosion of new sequence data has facilitated the use of comparative methods to understand group I intron evolution in a broader context and to generate hypotheses about intron insertion, splicing and spread that can be tested experimentally.     

Self-splicing of the Chlamydomonas chloroplast psbA introns.

We used alpha-32P-GTP labeling of total RNA preparations to identify self-splicing group I introns in Chlamydomonas. Several RNAs become labeled with alpha-32P-GTP, a subset of which is not seen with RNA from a mutant that lacks both copies of the psbA gene. Hybridization of the GTP-labeled RNAs to chloroplast DNA indicates that they originate from the psbA and rrn 23S genes, respectively, the only genes known to contain group I introns in this organism. Introns 1, 2, and 3 of psbA (with flanking exon sequences) were subcloned and transcribed in vitro. The synthetic RNAs were found to self-splice; splicing required Mg2+, GTP, and elevated temperature. In addition, the accuracy of self-splicing was confirmed for introns 1 and 2, and intermediates in the splicing reactions were detected. These results, together with our recent data on the 23S intron, indicate that the ability to self-splice is a general feature of Chlamydomonas group I introns. These findings have significant implications for the mechanism of group I intron splicing and evolution in Chlamydomonas and other chloroplast genomes.     

Self-splicing of the bacteriophage T4 group I introns requires efficient translation of the pre-mRNA in vivo and correlates with the growth state of the infected bacterium

Bacteriophage T4 contains three self-splicing group I introns in genes in de novo deoxyribonucleotide biosynthesis (in td, coding for thymidylate synthase and in nrdB and nrdD, coding for ribonucleotide reductase). Their presence in these genes has fueled speculations that the introns are retained within the phage genome due to a possible regulatory role in the control of de novo deoxyribonucleotide synthesis. To study whether sequences in the upstream exon interfere with proper intron folding and splicing, we inhibited translation in T4-infected bacteria as well as in bacteria containing recombinant plasmids carrying the nrdB intron. Splicing was strongly reduced for all three T4 introns after the addition of chloramphenicol during phage infection, suggesting that the need for translating ribosomes is a general trait for unperturbed splicing. The splicing of the cloned nrdB intron was markedly reduced in the presence of chloramphenicol or when translation was hindered by stop codons inserted in the upstream exon. Several exon regions capable of forming putative interactions with nrdB intron sequences were identified, and the removal or mutation of these exon regions restored splicing efficiency in the absence of translation. Interestingly, splicing of the cloned nrdB intron was also reduced as cells entered stationary phase and splicing of all three introns was reduced upon the T4 infection of stationary-phase bacteria. Our results imply that conditions likely to be frequently encountered by natural phage populations may limit the self-splicing efficiency of group I introns. This is the first time that environmental effects on bacterial growth have been linked to the regulation of splicing of phage introns.     

A molecular beacon assay for monitoring RNA splicing

Small molecule targeting of self-splicing RNAs like group I and II introns has been limited in part by the lack of a universal high-throughput screening platform for studies of splicing inhibition and kinetics. Here, we present the development of a molecular beacon assay for monitoring the accumulation of spliced exons during RNA splicing reactions. In this case, we applied it to the autocatalyzed reaction of the H.c.LSU group II intron found in the mitochondria of the pathogenic dimorphic fungus Histoplasma capsulatum. We find that a molecular beacon with the loop length of 18 nucleotides selectively recognizes ligated exons formed during self-splicing and exhibits high fluorescent signal upon binding of its target. We demonstrate that the fluorescent assay using molecular beacons can be successfully applied to kinetic characterization of the splicing reaction and determination of inhibition constants for small molecules. The results presented herein offer support for a molecular beacon approach to identifying small molecule inhibitors of intron splicing.     

A maturase-encoding group IIA intron of yeast mitochondria self-splices in vitro.

Intron 1 of the coxI gene of yeast mitochondrial DNA (aI1) is a group IIA intron that encodes a maturase function required for its splicing in vivo. It is shown here to self-splice in vitro under some reaction conditions reported earlier to yield efficient self-splicing of group IIB introns of yeast mtDNA that do not encode maturase functions. Unlike the group IIB introns, aI1 is inactive in 10 mM Mg2+ (including spermidine) and requires much higher levels of Mg2+ and added salts (1M NH4Cl or KCl or 2M (NH4)2SO4) for ready detection of splicing activity. In KCl-stimulated reactions, splicing occurs with little normal branch formation; a post-splicing reaction of linear excised intron RNA that forms shorter lariat RNAs with branches at cryptic sites was evident in those samples. At low levels of added NH4Cl or KCl, the precursor RNA carries out the first reaction step but appears blocked in the splicing step. AI1 RNA is most reactive at 37-42 degrees C, as compared with 45 degrees C for the group IIB introns; and it lacks the KCl- or NH4Cl-dependent spliced-exon reopening reaction that is evident for the self-splicing group IIB introns of yeast mitochondria. Like the group IIB intron aI5 gamma, the domain 4 of aI1 can be largely deleted in cis, without blocking splicing; also, trans-splicing of half molecules interrupted in domain 4 occurs. This is the first report of a maturase-encoding intron of either group I or group II that self-splices in vitro.     

RNA splicing in Neurospora mitochondria. Defective splicing of mitochondrial mRNA precursors in the nuclear mutant cyt18-1

cyt18-1 (299-9) is a nuclear mutant of Neurospora crassa that has been shown to have a temperature-sensitive defect in splicing the mitochondrial large rRNA intron. In the present work, we investigate the effect of the cyt18-1 mutation on splicing of mitochondrial mRNA introns. Two genes were studied in detail; the cytochrome b (cob) gene, which contains two introns, and a "long form" of the cytochrome oxidase subunit I (coI) gene, which contains four introns. We found that splicing of both cob introns and splicing of at least two of the coI introns are strongly inhibited in the mutant, whereas splicing of coI intron 1, which is excised as a 2.6 X 10(3) base circle, is relatively unaffected. The rRNA intron and both cob introns are group I introns, whereas the circular coI intron may belong to another structural class. Control experiments showed that the degree of inhibition of splicing is greater in the mutant than can be accounted for by severe inhibition of mitochondrial protein synthesis. Finally, experiments in which mutant cells were shifted from 25 degrees C to 37 degrees C showed that splicing of the large rRNA precursor and splicing of the coI mRNA precursor are inhibited with similar kinetics. Considered together, our results suggest that the cyt18 gene encodes a trans-acting component that is required for the splicing of group I mitochondrial DNA introns or some subclass thereof. Since Neurospora cob intron 1 has been shown to be self-splicing in vitro, defective splicing of this intron in cyt18-1 indicates that an essentially RNA-catalyzed splicing reaction must be facilitated by a trans-acting factor, presumably a protein, in vivo.     

A unique group of self-splicing introns in bacteriophage T4

We describe in this review, the salient splicing features of group I introns of bacteriophage T4 and propose, a hypothetical model to fit in the self-splicing of nrdB intron of T4 phage. Occurrence of non-coding sequences in prokaryotic cells is a rare event while it is common in eukaryotic cells, especially the higher eukaryotes. Therefore, T4 bacteriophage can serve as a good model system to study the evolutionary aspects of splicing of introns. Three genes of T4 phage were found to have stretches of non-coding sequences which belonged to the group IA type introns of self-splicing nature.     

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.     

Activity and thermostability of the small self-splicing group I intron in the pre-tRNA(lle) of the purple bacterium Azoarcus.

The 205-nt group I intron located in the pre-tRNA(lle) from the bacterium Azoarcus sp.BH72 is the smallest self-splicing group I intron identified to date. Comparative sequence analysis has placed this intron and the Anabaena pre-tRNA(Leu) intron into the same subgroup, IC3; we now compare their activity and stability. Unlike the Anabaena intron, the Azoarcus intron has two transitions in the kinetics of the first step of splicing. The faster transition occurs with a larger k(cat)/K(m) than that of the Anabaena or other group I introns, due to a rapid K(cat) (5 min(-1) at 32 degrees C) and a low K(m) for guanosine (17 microM). The excised intron circularizes by releasing a trinucleotide from the 5' end of the intron, another property unlike the Anabaena intron. Although it is smaller in size, the Azoarcus intron retains activity at higher temperatures, higher concentrations of urea, and higher pH than the Anabaena intron. Melting curves show that tertiary structure is disrupted at a lower temperature in the Anabaena intron. Some structural features that may explain the unusual stability of the Azoarcus intron include a G-C rich secondary structure and the presence of two 11-nt motifs, which are known to interact strongly with GAAA loops in group I and group II introns. The disruption of one of these interactions by substituting the Anabaena structural element in fact lowered the thermal stability of the Azoarcus intron. Thus, even superficially similar group I introns from the same structural subgroup can differ significantly in activity and stability.     

Excised group II introns in yeast mitochondria are lariats and can be formed by self-splicing in vitro

Excised group II introns in yeast mitochondria appear as covalently closed circles under the electron microscope. We show that these circular molecules are branched and resemble the lariats arising through splicing of nuclear pre-mRNAs in yeast and higher eukaryotes. One member of this intron class (aI5c in the gene for cytochrome c oxidase subunit I) is capable of self-splicing in vitro, giving correct exon-exon ligation and resulting in the appearance of both linear and lariat forms of the excised intron. Nuclease digestion of the latter molecules reveals the presence of a complex oligonucleotide with the probable structure AGU, which thus resembles the branch point formed in the spliceosome-dependent reactions undergone by nuclear pre-mRNAs. Unlike group I introns, this group II intron is not demonstrably dependent on GTP for self-splicing and circularization of the isolated, linear intron is not observed. A model accounting for these observations is presented.     

Rare group I intron with insertion sequence element in a bacterial ribonucleotide reductase gene

A rare group I intron in a cyanobacterial ribonucleotide reductase gene has been characterized. It contains a mobile insertion sequence element not required for RNA splicing. Ribonucleotide reductase genes were found to be hot spots for all three types of self-splicing intervening sequences, including group I and II introns and inteins.     

CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron.

The terminal intron (bI2) of the yeast mitochondrial cytochrome b gene is a group I intron capable of self-splicing in vitro at high concentrations of Mg2+. Excision of bI2 in vivo, however, requires a protein encoded by the nuclear gene CBP2. The CBP2 protein has been partially purified from wild-type yeast mitochondria and shown to promote splicing at physiological concentrations of Mg2+. The self-splicing and protein-dependent splicing reactions utilized a guanosine nucleoside cofactor, the hallmark of group I intron self-splicing reactions. Furthermore, mutations that abolished the autocatalytic activity of bI2 also blocked protein-dependent splicing. These results indicated that protein-dependent excision of bI2 is an RNA-catalyzed process involving the same two-step transesterification mechanism responsible for self-splicing of group I introns. We propose that the CBP2 protein binds to the bI2 precursor, thereby stabilizing the catalytically active structure of the RNA. The same or a similar RNA structure is probably induced by high concentrations of Mg2+ in the absence of protein. Binding of the CBP2 protein to the unspliced precursor was supported by the observation that the protein-dependent reaction was saturable by the wild-type precursor. Protein-dependent splicing was competitively inhibited by excised bI2 and by a splicing-defective precursor with a mutation in the 5' exon near the splice site but not by a splicing-defective precursor with a mutation in the core structure. Binding of the CBP2 protein to the precursor RNA had an effect on the 5' splice site helix, as evidenced by suppression of the interaction of an exogenous dinucleotide with the internal guide sequence.