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.     

Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification

The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides—some containing exons—were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3′ overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules.     

The pseudodisaccharides: a novel class of group I intron splicing inhibitors.

Lysinomicin, a naturally-occurring pseudodisaccharide, inhibits translation in prokaryotes. We report that lysinomicin (and three related compounds) are able to inhibit the self-splicing of group I introns, thus identifying pseudodisaccharides as a novel class of group I intron splicing inhibitors. Lysinomicin inhibited the self-splicing of the sunY intron of phage T4 with a Ki of 8.5 microM (+/- 5 microM) and was active against other group I introns. Inhibition was found to be competitive with the substrate guanosine, unlike aminoglycoside antibiotics, which act non-competitively to inhibit the splicing of group I introns. Competitive inhibitors of group I intron splicing known to date all contain a guanidino group that was thought to be required for inhibition; lysinomicin lacks a guanidino group.     

Activity of chimeric RNAs of U6 snRNA and (-)sTRSV in the cleavage of a substrate RNA.

U6 small nuclear RNA is one of the spliceosomal RNAs essential for pre-mRNA splicing. Discovery of mRNA-type introns in the highly conserved region of the U6 snRNA genes led to the hypothesis that U6 snRNA functions as a catalytic element during pre-mRNA splicing. The highly conserved region of U6 snRNA has a structural similarity with the catalytic domain of the negative strand of the satellite RNA of tobacco ring spot virus [(-)sTRSV], suggesting that the highly conserved region of U6 snRNA forms the catalytic center. We examined whether synthetic RNAs consisting of the sequence of the highly conserved region of U6 snRNA or various chimeric RNAs between the U6 region and the catalytic RNA of (-)sTRSV could cleave a substrate RNA that can partially base-pair with them and have a GU sequence. Chimeric RNAs with 70 to 83% sequence identity with the conserved region of S. pombe U6 snRNA cleaved the substrate RNA at the 5' side of the GU sequence, which is shared by the 5' end of an intron in a pre-mRNA. We found that the highly conserved region of U6 snRNA and the catalytic domain of (-)sTRSV are strikingly similar in structure to the catalytic core region of the group I self-splicing intron in cyanobacteria. These results suggest that U6 snRNA, (-)sTRSV and the group I self-splicing intron originated from a common ancestral RNA, and support the hypothesis that U6 snRNA catalyzes pre-mRNA splicing reaction.     

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.     

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 common group I intron between a plant parasitic fungus and its host

The self-splicing RNAs known as group I introns exist in many organisms, but their distribution is difficult to explain. We hypothesize that group I introns have been transferred between a parasite and its host. We describe here the discovery of a common group I intron sequence between a plant-parasitic fungus, Protomyces inouyei, and its host, Youngia japonica. It strongly supports our theory that the group I intron had been transferred from the host plant to the parasitic fungus in the course of evolution.      

In vitro characterization of the splicing efficiency and fidelity of the RmInt1 group II intron as a means of controlling the dispersion of its host mobile element

Group II introns are catalytic RNAs that are excised from their precursors in a protein-dependent manner in vivo. Certain group II introns can also react in a protein-independent manner under nonphysiological conditions in vitro. The efficiency and fidelity of the splicing reaction is crucial, to guarantee the correct formation and expression of the protein-coding mRNA. RmInt1 is an efficient mobile intron found within the ISRm2011-2 insertion sequence in the symbiotic bacterium Sinorhizobium meliloti. The RmInt1 intron self-splices in vitro, but this reaction generates side products due to a predicted cryptic IBS1* sequence within the 3′ exon. We engineered an RmInt1 intron lacking the cryptic IBS1* sequence, which improved the fidelity of the splicing reaction. However, atypical circular forms of similar electrophoretic mobility to the lariat intron were nevertheless observed. We analyzed a run of four cytidine residues at the 3′ splice site potentially responsible for a lack of fidelity at this site leading to the formation of circular intron forms. We showed that mutations of residues base-pairing in the tertiary EBS3–IBS3 interaction increased the efficiency and fidelity of the splicing reaction. Our results indicate that RmInt1 has developed strategies for decreasing its splicing efficiency and fidelity. RmInt1 makes use of unproductive splicing reactions to limit the transposition of the insertion sequence into which it inserts itself in its natural context, thereby preventing potentially harmful dispersion of ISRm2011-2 throughout the genome of its host.     

Group I permuted intron-exon (PIE) sequences self-splice to produce circular exons.

Circularly permuted group I intron precursor RNAs, containing end-to-end fused exons which interrupt half-intron sequences, were generated and tested for self-splicing activity. An autocatalytic RNA can form when the primary order of essential intron sequence elements, splice sites, and exons are permuted in this manner. Covalent attachment of guanosine to the 5' half-intron product, and accurate exon ligation indicated that the mechanism and specificity of splicing were not altered. However, because the exons were fused and the order of the splice sites reversed, splicing released the fused-exon as a circle. With this arrangement of splice sites, circular exon production was a prediction of the group I splicing mechanism. Circular RNAs have properties that would make them attractive for certain studies of RNA structure and function. Reversal of splice site sequences in a context that allows splicing, such as those generated by circularly permuted group I introns, could be used to generate short defined sequences of circular RNA in vitro and perhaps in vivo.     

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.     

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.     

Metal binding and substrate positioning by evolutionarily invariant U6 sequences in catalytically active protein-free snRNAs

We have previously shown that a base-paired complex formed by two of the spliceosomal RNA components, U6 and U2 small nuclear RNAs (snRNAs), can catalyze a two-step splicing reaction that depended on an evolutionarily invariant region in U6, the ACAGAGA box. Here we further analyze this RNA-catalyzed reaction and show that while the 5′ and 3′ splice site substrates are juxtaposed and positioned near the ACAGAGA sequence in U6, the role of the snRNAs in the reaction is beyond mere juxtaposition of the substrates and likely involves the formation of a sophisticated active site. Interestingly, the snRNA-catalyzed reaction is metal dependent, as is the case with other known splicing RNA enzymes, and terbium(III) cleavage reactions indicate metal binding by the U6/U2 complex within the evolutionarily conserved regions of U6. The above results, combined with the structural similarities between U6 and catalytically critical domains in group II self-splicing introns, suggest that the base-paired complex of U6 and U2 snRNAs is a vestigial ribozyme and a likely descendant of a group II-like self-splicing intron.     

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.     

Updated Progress on Group II Intron Splicing Factors in Plant Chloroplasts

Group II introns are large catalytic RNAs (ribozymes) in the bacteria and organelle genomes of several lower eukaryotes. Many critical photosynthesis-related genes in the plant chloroplast genome also contain group II introns, and their splicing is critical for chloroplast biogenesis and photosynthesis processes. The structure of chloroplast group II introns was altered during evolution, resulting in the loss of intron self-splicing. Therefore, the assistance of protein factors was required for their splicing processes. As an increasing number of studies focus on the mechanism of chloroplast intron splicing; many new nuclear-encoded splicing factors that are involved in the chloroplast intron splicing process have been reported. This report reviewed the research progress of the updated splicing factors found to be involved in the splicing of chloroplast group II introns. We discuss the main problems that remain in this research field and suggest future research directions.     

Mutagenesis of a light-regulated psbA intron reveals the importance of efficient splicing for photosynthetic growth

The chloroplast-encoded psbA gene encodes the D1 polypeptide of the photosystem II reaction center, which is synthesized at high rates in the light. In Chlamydomonas reinhardtii, the psbA gene contains four self-splicing group I introns whose rates of splicing in vivo are increased at least 6–10-fold by light. However, because psbA is an abundant mRNA, and some chloroplast mRNAs appear to be in great excess of what is needed to sustain translation rates, the developmental significance of light-promoted splicing has not been clear. To address this and other questions, potentially destabilizing substitutions were made in several predicted helices of the fourth psbA intron, Cr.psbA4, and their effects on in vitro and in vivo splicing assessed. Two-nucleotide substitutions in P4 and P7 were necessary to substantially reduce splicing of this intron in vivo, although most mutations reduced self-splicing in vitro. The P7-4,5 mutant, whose splicing was completely blocked, showed no photoautotrophic growth and synthesis of a truncated D1 (exons 1–4) polypeptide from the unspliced mRNA. Most informative was the P4′-3,4 mutant, which exhibited a 45% reduction in spliced psbA mRNA, a 28% reduction in synthesis of full-length D1, and an 18% reduction in photoautotrophic growth. These results indicate that psbA mRNA is not in great excess, and that highly efficient splicing of psbA introns, which is afforded by light conditions, is necessary for optimal photosynthetic growth.     

RNA splicing in the T-even bacteriophage

Group 1 introns, first demonstrated in the nuclear large rRNA of Tetrahymena thermophila and subsequently in many yeast, fungal mitochondrial, and chloroplast precursor RNAs, are capable of intron excision and exon ligation in vitro, although this process occurs much more rapidly in vivo. The discovery and characterization of a similar intron in the T4 phage thymidylate synthase gene (td) led to the finding of additional group 1 introns in other T4 genes and in genes of the related T2 and T6 phages. Because protein factors are not required in the splicing of group 1 introns in vitro, it has been postulated that the precursor RNA can assume a critical conformation enabling it to undergo site-specific autocatalytic cleavage and ligation (self-splicing). By means of site-directed mutation, it has been shown unequivocally that several sequence elements in the Tetrahymena rRNA intron are involved in the formation of base-paired stem structures that are essential for the self-splicing process. These sequence elements have been demonstrated in other eukaryotic group 1 introns, as well as in the td intron. In this brief review we shall describe the biochemical and structural properties of the td intron in relation to other newly found phage introns. The interesting implications arising from these revelations will also be discussed.