Circle reopening in the Tetrahymena ribozyme resembles site-specific hydrolysis at the 3' splice site.

The Tetrahymena intron, after splicing from its flanking exons, can mediate its own circularization. This is followed by site-specific hydrolysis of the phosphodiester bond formed during the circularization reaction. The structural components involved in recognition of this bond for hydrolysis have not been established. We have made base substitutions to the P9.0 pairing and at the 3'-terminal guanosine residue (G414) of the intron to investigate their effects on circle formation and reopening. We have found that disruption of either P9.0 pairing or binding of the terminal nucleotide result in the formation of a large circle, C-413:5E23 from precursor RNA molecules that have undergone hydrolysis at the 3' splice site. This circle is formed at the phosphodiester bond of the 5'-terminal guanosine residue of the upstream exon via nucleophilic attack by the 3'-terminal nucleotide of the intron. The large circle is novel since it can reopen eight bases downstream from the original circularization junction at a site resembling the normal 3' splice site, restoring a guanosine to the 3' terminus and re-establishing P9.0 pairing. The new 3' terminus of the intron is capable of recircularization at any of the three normal wild-type sites. We conclude that both P9.0 and the 3'-terminal guanosine residue are required for the selection of the phosphodiester bond hydrolysed during circle reopening.     

Characterization of products derived from self-splicing of intron aI5 alpha which is located in the mitochondrial COX I gene of Saccharomyces cerevisiae.

We have characterized the in vitro self-splicing of intron aI5 alpha containing precursor RNA from the yeast mitochondrial gene coding for cytochrome oxidase subunit I. This intron follows the rules for group I self-splicing introns and all the characteristic products have been identified. In addition we have detected abnormal RNA products with features that indicate that the self-splicing behaviour of this intron is more complex. Two intron circles are formed by use of a major and minor intron-internal site for circle closure. A cryptic 5'-splice site located in the 3' exon results in guanosine nucleotide mediated opening at a position 30 nt downstream of the normal 3' splice site. The reactions can all be explained on the basis of the "splice guide" model proposed by Davies et al (1982 Nature 300 719-724). Although the sequence motifs at cyclization and splice sites occur more often in this intron, only some of them are allowed to interact with the internal guide sequence, suggesting that both primary structure and spatial folding of the RNA are involved in formation of productive reaction sites.     

Guanosine binding required for cyclization of the self-splicing intervening sequence ribonucleic acid from Tetrahymena thermophila

We have converted the intramolecular cyclization reaction of the self-splicing intervening sequence (IVS) ribonucleic acid (RNA) from Tetrahymena thermophila into an intermolecular guanosine addition reaction. This was accomplished by selectively removing the 3'-terminal nucleotide by oxidation and beta-elimination; the beta-eliminated IVS thereby is no longer capable of reacting with itself. However, under cyclization conditions, a free guanosine molecule can make a nucleophilic attack at the normal cyclization site. We have used this guanosine addition reaction as a model system for a Michaelis-Menten kinetic analysis of the guanosine binding site involved in cyclization. The results indicate that functional groups on the guanine that are used in a G-C Watson-Crick base pair are important for the cyclization reaction. This is the same result that was obtained for the guanosine binding site involved in splicing [Bass, B. L., & Cech, T. R. (1984) Nature (London) 308, 820-826]. Unlike splicing, however, certain additional nucleotides 5' to the guanosine moiety make significant binding contributions. We conclude that the guanosine binding site in cyclization is similar to, but not identical with, the guanosine binding site in splicing. The same binding interactions used in cyclization could help align the 3' splice site of the rRNA precursor for exon ligation. We also report that the phosphodiester bond at the cyclization site is susceptible to a pH-dependent hydrolysis reaction; the phosphodiester bond is somehow activated toward attack by the 3'hydroxyl of a guanosine molecule or by a hydroxyl ion.     

Novel RNA polymerization reaction catalyzed by a group I ribozyme.

We have converted a bacterial tRNA precursor containing a 205 nt self-splicing group I intron into a RNA enzyme that catalyzes polymerization of an external RNA substrate. The reaction involves transesterification steps analogous to both the forward and reverse exon ligation steps of group I splicing; as such it depends entirely on 3' splice site reactions. The RNA substrate is a 20 nt analogue of the ligated exons (E1.E2), whose 3' end resembles the 3' terminus of the intron RNA enzyme (IVS). The splice junction of the substrate is attacked by the 3' end of the intron, then the molecule displaces the original 3' terminal guanosine so that the new 3' terminus is brought into the active site and used as the attacking nucleophile in the next reaction. Polymerization occurs via a series of covalent enzyme-linked intermediates of the structure IVS.(E2)n, where n = 1 to > or = 18. The 5' exon accumulates during the course of the reaction and can attack the covalent intermediates to produce elongation products of structure E1.(E2)n, regenerating the intron RNA enzyme in unchanged form. In this manner, the enzyme converts 20 nt oligoribonucleotides into polyribonucleotides up to at least 180 nt by 10 nt increments. These results have significant implications for the evolution of RNA-based self-replicating systems.     

Ribozyme inhibitors: deoxyguanosine and dideoxyguanosine are competitive inhibitors of self-splicing of the Tetrahymena ribosomal ribonucleic acid precursor

The intervening sequence (IVS) of the Tetrahymena rRNA precursor catalyzes its own splicing. During splicing the 3'-hydroxyl of guanosine is ligated to the 5' terminus of the IVS. One catalytic strategy of the IVS RNA is to specifically bind its guanosine substrate. Deoxyguanosine (dG) and dideoxyguanosine (ddG) are found to be competitive inhibitors of self-splicing. Comparison of the kinetic parameters (Ki = 1.1 mM for dG; Ki = 5.4 mM for ddG; Km = 0.032 mM for guanosine) indicates that the ribose hydroxyls are necessary for optimal binding of guanosine to the RNA. dG is not a substrate for the reaction even at very high concentrations. Thus, in addition to aiding in binding, the 2'-hydroxyl is necessary for reaction of the 3'-hydroxyl. A second catalytic strategy of the IVS RNA is to enhance the reactivity of specific bonds. For example, the phosphodiester bond at the 3' splice site is extremely labile to hydrolysis. We find that dG and ddG, as well as 2'-O-methylguanosine and 3'-O-methylguanosine, reduce hydrolysis at the 3' splice site. These data are consistent with an RNA structure that brings the 5' and 3' splice sites proximal to the guanosine binding site.     

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.     

Reactions of the intervening sequence of the Tetrahymena ribosomal ribonucleic acid precursor: pH dependence of cyclization and site-specific hydrolysis

During self-splicing of the Tetrahymena rRNA precursor, the intervening sequence (IVS) is excised as a unique linear molecule and subsequently cyclized. Cyclization involves formation of a phosphodiester bond between the 3' end and nucleotide 16 of the linear RNA, with release of an oligonucleotide containing the first 15 nucleotides. We find that the rate of cyclization is independent of pH in the range 4.7-9.0. A minor site of cyclization at nucleotide 20 is characterized. Cyclization to this site becomes more prominent at higher pHs, although under all conditions examined it is minor compared to cyclization at nucleotide 16. The circular IVS RNAs are unstable, undergoing hydrolysis at the phosphodiester bond that was formed during cyclization. We find that the rate of site-specific hydrolysis is first order with respect to hydroxide ion concentration, with a rate constant 10(3)-10(4)-fold greater than that of hydrolysis of strained cyclic phosphate esters. On the basis of these results, we propose that circular IVS RNA hydrolysis involves direct attack of OH- on the phosphate at the ligation junction, that particular phosphate being made particularly reactive by the folding of the RNA molecule. Cyclization, on the other hand, appears to occur by direct attack of the 3'-terminal hydroxyl group of the linear IVS RNA without prior deprotonation.     

Stereochemical selectivity of group II intron splicing, reverse splicing, and hydrolysis reactions.

We have previously shown, using phosphorothioate substitutions at splice site, that both transesterification steps of group II intron self-splicing proceed, by stereochemical inversion, with an Sp but not an Rp phosphorothioate. Under alternative reaction conditions or with various intron fragments, group II introns can splice following hydrolysis at the 5' splice site and can also hydrolyze the bond between spliced exons (the spliced-exon reopening reaction). In this study, we have determined the stereochemical specificities of all of the major model hydrolytic reactions carried out by the aI5 gamma intron from Saccharomyces cerevisiae mitochondria. For all substrates containing exon 1 and most of the intron, the stereospecificity of hydrolysis is the same as for the step 1 transesterification reaction. In contrast, the spliced-exon reopening reaction proceeds with an Rp but not an Sp phosphorothioate at the scissile bond, as does true reverse splicing. Thus, by stereochemistry, this reaction appears to be related to the reverse of step 2 of self-splicing. Finally, a substrate RNA that contains the first exon and nine nucleotides of the intron, when reacted with the intron ribozyme, releases the first exon regardless of the configuration of the phosphorothioate at the 5' splice site, suggesting that this substrate can be cleaved by either the step 1 or the step 2 reaction site. Our findings clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system and permit new studies to be interpreted more rigorously.     

Cryptic branch point activation allows accurate in vitro splicing of human beta-globin intron mutants

The excised introns of pre-mRNAs and intron-containing splicing intermediates are in a lariat configuration in which the 5' end of the intron is linked by a 2'-5' phosphodiester bond (RNA branch) to a single adenosine residue near the 3' end of the intron. To determine the role of the specific sequence surrounding the RNA branch, we have mutated the branch point sequence of the human beta-globin IVS1. Pre-mRNAs lacking the authentic branch point sequence are accurately spliced in vitro; processing of the mutant pre-mRNAs generates RNA lariats due to the activation of cryptic branch points within IVS1. The cryptic branch points always occur at adenosine residues, but the sequences surrounding the branched nucleotide vary. Regardless of the type of mutation or the sequences remaining within IVS1, the cryptic branch points are 22 to 37 nucleotides upstream of the 3' splice site. These results suggest that RNA branch point selection is primarily based on a mechanism that measures the distance from the 3' splice site.     

Purification of a RNA debranching activity from HeLa cells

The splicing of messenger RNA precursors (pre-mRNA) of eukaryotic cells involves the formation of a branched RNA intermediate known as a RNA lariat. This structure is formed in the first step of the reaction when a cleavage at the 5' splice site generates the 5' exon and a RNA species containing the intron and 3' exon in which the phosphate moiety at the 5' end of the intron is forming a 2'-5' phosphodiester bond with the 2'-hydroxyl moiety of a specific adenine residue near the 3' end of the intron forming a RNA branch with the following structure: -pA2'-pX-3'-pZ-. We have purified a debranching activity approximately 700-fold from the cytosolic fraction of HeLa cells. This activity catalyzes the hydrolysis of the 2'-5' phosphodiester bond of branched RNA structures yielding a 5'-phosphate end and a 2'-hydroxyl group at the branch attachment site. The activity possessed a sedimentation coefficient of 3.5 S. The reaction catalyzed by the purified fraction requires a divalent cation and is optimal at pH 7.0. The purified activity can efficiently hydrolyze triester trinucleotide structures (pY2'-pX-3'-pZ-) prepared by digestion of RNA lariats with nuclease P1. In contrast, a 2' phosphate monoester product (-pG2'-p 3'-pC-), formed by the wheat germ RNA ligase, was not attacked.     

Group II introns deleted for multiple substructures retain self-splicing activity.

Group II introns can be folded into highly conserved secondary structures with six major substructures or domains. Domains 1 and 5 are known to play key roles in self-splicing, while the roles of domains 2, 3, 4, and 6 are less clear. A trans assay for domain 5 function has been developed which indicates that domain 5 has a binding site on the precursor RNA that is not predicted from any secondary structure element. In this study, the self-splicing group II intron 5 gamma of the coxI gene of yeast mitochondrial DNA was deleted for various intron domains, singly and in combinations. Those mutant introns were characterized for self-splicing reactions in vitro as a means of locating the domain 5 binding site. A single deletion of domain 2, 3, 4, or 6 does not block in vitro reactions at either splice junction, though the deletion of domain 6 reduces the fidelity of 3' splice site selection somewhat. Even the triple deletion lacking domains 2, 4, and 6 retains some self-splicing activity. The deletion of domains 2, 3, 4, and 6 blocks the reaction at the 3' splice junction but not at the 5' junction. From these results, we conclude that the binding site for domain 5 is within domain 1 and that the complex of 5' exon, domain 1, and domain 5 (plus short connecting sequences) constitutes the essential catalytic core of this intron.     

Reactivity of modified ribose moieties of guanosine: new cleavage reactions mediated by the IVS of Tetrahymena precursor rRNA.

An RNA molecule consisting of the 5' exon and intervening sequence (IVS) of Tetrahymena precursor rRNA was oxidized with sodium periodate to convert the ribose moiety of the 3' terminal guanosine into a dialdehyde form. The modified RNA undergoes a specific cleavage reaction at the 5' splice site, but has no apparent cyclization activity. This novel reaction mediated by the IVS RNA is pH dependent over the range 6.5-8.5 and leaves a 5' phosphate and a 3'-OH at the newly created termini. The dialdehyde form of monomer guanosine is also capable of causing a specific cleavage reaction at the 5' splice site although the nucleotide is not covalently attached to the IVS RNA in the final product. These and other findings described in this report suggest that the cis diol of the intact ribose moiety of guanosine is not an absolute requirement for the IVS-mediated reactions.     

Formation of lariats and circles in self-splicing of the precursor to the large ribosomal RNA of yeast mitochondria

Self-splicing of the precursor to large ribosomal RNA of yeast mitochondria leads not only to circles but also to lariats, structures that have not been observed before as products of self-splicing. Lariats were studied by electron microscopy after hybridization with an RNA complementary to the 3' half of the precursor. This leads to differentiation in at least two classes of lariats that vary in the position of the branch point. In all lariats the tail carries the 3' end, which suggests that a 5' end is used for branch formation with an internal nucleotide. The circles are formed from excised introns. They lack only three nucleotides encoded by mitochondrial DNA along with the 5'-terminal G added in the course of self-splicing. The diverse number of self-splicing products arising in vitro testifies to the considerable reactivity of this intron. The formation of lariats in an RNA catalyzed reaction may have implications for views on the mechanism of splicing of nuclear pre-mRNAs.     

Molecular recognition in a trans excision-splicing ribozyme: non-Watson-Crick base pairs at the 5' splice site and omegaG at the 3' splice site can play a role in determining the binding register of reaction substrates

Trans excision-splicing (TES) ribozymes, derived from a Pneumocystis carinii group I intron, can catalyze the excision of targeted sequences from within RNAs. In this report, the sequence requirements of the splice sites are analyzed. These conserved sequences include a u-G wobble pair at the 5' splice site and a guanosine in the omega position at the 3' splice site (in the substrate). We report that 7 out of 16 base pair combinations at the 5' splice site produce appreciable TES product. This promiscuity is in contrast to results reported for analogous self-splicing reactions using a Tetrahymena ribozyme. At long reaction times TES products dissociate and rebind free ribozyme, at which point product degradation occurs via the 5' cleavage reaction. Unexpectedly, only in cases where Watson-Crick base pairs form at the 5'splice site do we see degradation of TES products at cryptic sites, suggesting that non-Watson-Crick base pairs at the 5' splice site are acting in concert with other factors to precisely determine the binding register of TES reaction substrates within the ribozyme. Moreover, cryptic site degradation does not occur with the corresponding reaction substrates, which additionally contain omegaG, suggesting that omegaG can play a similar role. We report that omegaG cannot be replaced by any other base, so TES substrates require a guanosine as the last (or only) base to be excised. Additionally, we demonstrate that P9.0 and P10 are expendable for TES reactions, suggesting that omegaG is sufficient as a 3' molecular recognition element.     

Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro

We have altered the TACTAAC sequence in the yeast CYH2m gene intron to TACTACC. This mutation changes the nucleotide at the normal position of the branch in intron RNA lariats produced during pre-mRNA splicing, and it prevents splicing in vivo. In a yeast pre-mRNA splicing system, CYH2m pre-mRNA carrying the TACTACC mutation is not specifically cut or rearranged in any way. Substitution of an A for the first G of the CYH2m intron, converting the highly conserved GTATGT 5' splice site sequence to ATATGT, also blocks intron excision in vivo and in vitro: pre-mRNA carrying this mutation was still cut normally at the mutant 5' splice site in vitro, to give authentic exon 1 and an intron-exon 2 lariat RNA with an A-A 2'-5' phosphodiester linkage at the branch point. This lariat RNA is a dead-end product. The subsequent cleavage at the 3' splice site is therefore sensitive to the sequence of the 5' end of the intron attached at the branch point.