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A review of the data on the synthesis of RNA-fragments with endonuclease activity. The Haseloff and Gerlach model for design of ribozymes and the results of the synthesis of the three new ribozymes targeted against the chloramphenicol acetyl transferase gene transcript are considered. The possibility of the use of a shortened form of Tetrahymena self-splicing ribosomal RNA intervening sequence for the ribozyme design also are discussed.
Mosaic structure of genes is shown for three out of four known types of RNA: transfer, ribosomal and messenger. At least three different mechanisms are involved in maturation of transcripts from these genes. The peculiarity of tRNA splicing is connected with the possibility of existence of tRNA molecules free of ribonucleoprotein complexes. The mode of Tetrahymena pre-tRNA self-splicing may also take place during maturation of other RNAs (rRNA of protozoa, ribosomal and messenger rRNA of lower fungi, plant messenger rRNA), which share the similar structure. The third type of mechanism is involved in splicing messenger RNA in eukaryotic cell nuclei.
The catalytic activity of ribonucleic acid is reviewed, with the intervening sequence (IVS) of the ribosomal RNA precursor of Tetrahymena serving as a major example. The IVS catalyzes its own excision from the precursor RNA and at the same time ligation of the flanking sequences, a reaction termed self-splicing. The excised IVS RNA can act as an enzyme to catalyze sequence-specific cleavage and ligation reactions on substrate RNA molecules. The RNA polymerization activity of the IVS supports the possibility that RNA catalysis could have been important in establishing a prebiotic self-replicating system. Other systems in which RNA catalysis has been found include related group I IVSs, group II IVSs, ribonuclease P, and certain plant infectious RNAs.
The 35 S percursor of the Neurospora mitochondrial large rRNA contains a 2.3-kilobase intron located towards its 3' end. The intron RNA is excised in a single cleavage-ligation reaction and is detectable in mitochondria by Northern hybridization experiments. We now show (i) that the free intron RNA is a full-length linear molecule, and (ii) that it, like the Tetrahymena nuclear rRNA intron, contains an extra, noncoded guanosine residue at its 5' end. The latter finding suggests that the Neurospora mitochondrial large rRNA may be spliced via a phosphoester transfer mechanism similar to that proposed for the "self-splicing" Tetrahymena intron.
The catalytic core of Group I self-splicing introns has been proposed to consist of two structural domains, P4-P6 and P3-P9. Each contains helical segments and conserved unpaired nucleotides, and the isolated P4-P6 domain has been shown to have substantial native tertiary structure. The proposed tertiary structure domains of the Tetrahymena intron were synthesized separately and shown to self-assemble into a catalytically active complex. Surprisingly, the concentration dependence of these reactions revealed that the domains interact with nanomolar apparent dissociation constants, even though there is no known base pairing between P4-P6 and P3-P9. This suggests that the domains interact through multiple tertiary contacts, the nature of which can now be explored in this system. For example, a circularly permuted version of the P4-P6 domain, which folds similarly to the native P4-P6 molecule, formed a stable but inactive complex. Interestingly, activity was demonstrated with the permuted molecule when nucleotides proposed to form a triple-strand interaction with P4 and P6 were restored as part of the P1-P3 substrate or as part of the P3-P9 RNA. Thus, beyond stabilization of the P4-P6 domain, the triple-strand region may facilitate correct orientation of the RNA domains or participate more directly in catalysis.
The observation that the large ribosomal RNA intron of Tetrahymena is spliced 20-50-fold more rapidly in vivo than in vitro (Brehm SL, Cech TR, 1983, Biochemistry 22:2390-2397; Bass BL, Cech TR, 1984, Nature 308:820-826) suggests facilitation of RNA folding in vivo. To determine whether a specific group I splicing factor is required in Tetrahymena, the intron was inserted into the analogous position of the Escherichia coli 23S rRNA. We report that the intron is rapidly excised from pre-rRNA in bacteria and that the magnitude of the in vivo rate enhancement is similar to that in Tetrahymena. These results demonstrate that a species-specific protein is not required. Instead, a common mechanism of assisting RNA folding is sufficient to accelerate the removal of self-splicing introns from ribosomal RNA.
The group II intron bl1, the first intron of the mitochondrial cytochrome b gene in yeast is self-splicing in vitro. Genetic evidence suggests that trans-acting factors are required for in vivo splicing of this intron. In accordance with these findings, we present in vitro data showing that splicing of bl1 under physiological conditions depends upon the presence of proteins of a mitochondrial lysate. ATP is an essential component in this reaction. Overexpression of the nuclear-encoded DEAD box protein pMSS116 results in a marked increase in the ATP-dependent splicing activity of the extract, suggesting that pMSS116 may play an important role in splicing of bl1.
The group II intron bI1, the first intron of the mitochondrial cytochrome b gene in yeast is self-splicing in vitro. Genetic evidence suggests that trans-acting factors are required for in vivo splicing of this intron. In accordance with these findings, we present in vitro data showing that splicing of bI1 under physiological conditions depends upon the presence of proteins of a mitochondrial lysate. ATP is an essential component is this reaction. Overexpression of the nuclear-encoded DEAD box protein pMSS-116 results in a marked increase in the ATP-dependent splicing activity of the extract, suggesting that pMSS116 may play an important role in splicing of bI1.
The function of the SunY protein, encoded by an intron-containing gene of bacteriophage T4, has remained hitherto unknown in contrast to the extensively studied self-splicing reaction of the SunY intron. Here we show that anaerobic T4 infections of Escherichia coli induce a ribonucleoside triphosphate reductase activity that is 10-30-fold higher than the bacterial host level of the corresponding enzyme. Inactivation of the T4 sunY gene (in this communication renamed nrdD) significantly decreased both the induced activity and the anaerobic production of phage, confirming the role of the T4 NrdD (SunY) protein as a phage-specific anaerobic ribonucleotide reductase. With the identification of the T4 nrdD (sunY) gene product as an anaerobic ribonucleotide reductase, all known bacteriophage introns are found to share the common and as yet unexplained property of residing within genes of DNA metabolism.
Circular exon sequences can be generated by splicing permuted intron-exon (PIE) sequences. The Anabaena pre-tRNA group I self-splicing PIE sequence was modified to generate circular forms of the HIV-TAR and the high affinity region of the HIV-RRE (RBE). RNA products containing TAR and the RBE were purified from splicing reactions and demonstrated to be circular. The circular form of these sequences was shown to be resistant to nuclease degradation in cellular extracts. Gel shift assays demonstrate that the circular form of the RBE is specifically bound by a Rev derived peptide. These data suggest that PIE-circularization of RNA may be an effective way to express small stable RNAs designed for therapeutics (eg-decoys).
Circular exon sequences can be generated by splicing permuted intron-exon (PIE) sequences. The Anabaena pre-tRNA group I self-splicing PIE sequence was modified to generate circular forms of the HIV-TAR and the high affinity region of the HIV-RRE (RBE). RNA products containing TAR and the RBE were purified from splicing reactions and demonstrated to be circular. The circular form of these sequences was shown to be resistant to nuclease degradation in cellular extracts. Gel shift assays demonstrate that the circular form of the RBE is specifically bound by a Rev derived peptide. These data suggest that PIE-circularization of RNA may be an effective way to express small stable RNAs designed for therapeutics (eg. decoys).
The kinetics of RNA folding were examined in the L-21 ribozyme, an RNA enzyme derived from the self-splicing Tetrahymena intron. Three UV-inducible crosslinks were mapped, characterized, and used as indicators for the folded state of the ribozyme. Together these data suggest that final structures are adopted first by the P4-P6 independently folding domain and only later in a region that positions the P1 helix (including the 5' splice site), a region whose folding is linked to that of a portion of the catalytic core. At intermediate times, a non-native structure forms in the region of the triple helical scaffold, which connects the major folding domains. At 30 degrees C, the unfolded ribozyme passes through these stages with a half-life of 2 min from the time magnesium cations are provided. At higher temperatures, the half-life is shortened but the order of events is unchanged. Thermal melting of the fully folded ribozyme also revealed a multi-stage process in which the steps of folding are reversed: the kinetically slowest structure is the least stable and melts first. These structures of the ribozyme also bind Mg2+ cooperatively and their relative affinity for binding seems to be a major determinant in the order of events during folding. Na+ can also substitute for Mg2+ to give rise to the same crosslinkable structures, but only at much higher concentrations. Specific binding sites for Mg2+ may make this cation particularly efficient at electrostatic stabilization during folding of these ribozyme structures.
The nucleotide sequences of two self-splicing group I introns found in viruses that infect the eukaryotic green alga Chlorella were compared with those of 19 nuclear group I introns of 11 green algae, 1 red alga, 2 yeasts and 1 protist. The similarity ranging from 69.5% to 89.0% in any comparison strongly suggested a close relatedness of these introns. Since this kind of viruses infect algal cells residing in protists such as Paramecium, they may serve as a mediator to spread group I introns horizontally among organisms of different phylogenetic kingdoms.
For small RNAs, isomorphous heavy-atom derivatives can be obtained by crystallizing synthetic versions that incorporate modified nucleotides such as iodo- or bromouridine. However, such a synthetic approach is not yet feasible for RNAs greater than approximately 40 nt. We have been investigating P4-P6, a 160-nt domain of the self-splicing Tetrahymena intron whose structure was solved recently (Cate JH et al., 1996, Science 273:1678-1685). To incorporate iodouridine, a two-piece RNA was constructed. The 5' segment, containing the majority of the molecule, was transcribed in vitro using a self-processing hammerhead ribozyme to cleave the nascent transcript and give a homogenous 3' end. A synthetic 5-iodouridine-containing RNA corresponding to the remainder of the sequence was then annealed to the transcribed piece of RNA. The resulting RNA appeared structurally and functionally sound as judged by nondenaturing gel electrophoresis and RNA cleavage assays. Four versions of this two-piece system with 5-iodouridine substitutions at different positions crystallized under the same conditions as the native RNA, yielding two useful heavy-atom derivatives of P4-P6. The position of the iodine atoms for the derivatives could be determined in the absence of phase information, and an interpretable electron density map was calculated using only the data from the two iodouridine derivatives. This approach is expected to be readily adaptable to other large, structured RNA molecules.
The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) functions in splicing the N. crassa mitochondrial large rRNA intron by stabilizing the catalytically active structure of the intron core. Here, a comprehensive study of N. crassa mtDNA group I introns identified two additional introns, cob-I2 and the ND1 intron, that are dependent on CYT-18 for splicing in vitro and in vivo. The other seven N. crassa mtDNA group I introns are not CYT-18-dependent and include five that self-splice and two that do not splice under any conditions examined. Some of these introns may require maturases or other proteins for efficient splicing. All but one of the non-CYT-18-dependent introns contain large peripheral extensions of the P5 stem, related to the P5abc structure that blocks CYT-18 binding to the Tetrahymena large rRNA intron. The remaining non-CYT-18-dependent intron, cob-I1, contains a long, peripheral extension of the P9 stem, denoted P9.1, which also impedes CYT-18 binding. Detailed analysis of the CYT-18-dependent ND1 intron showed that two 3' splice sites are used in vitro and in vivo. The proximal, alternative 3' splice site brings the intron open reading frame, which potentially encodes a mobility endonuclease, in frame with the upstream exon, possibly providing a means of expression. Considered together, our results show that group I introns in N. crassa mitochondria use a variety of strategies involving different proteins and/or RNA structures to assist splicing, and they support the hypothesis that CYT-18 and the peripheral RNA structure P5abc are alternative evolutionary adaptations for stabilizing the active structure of the intron core.
The precise excision of intervening sequences during RNA splicing is an interesting example of the high degree of specificity involved in biosynthesis processes. Self-splicing RNA precursors achieve this specificity primarily through intramolecular interactions whereas all other types of RNA splicing requires interaction between cellular factors and specific recognition signals in the RNA precursor. About twelve years ago, the in vitro splicing system was developed and a general scheme of the pre-mRNA was proposed (Hernandez and Keller, 1983; Krainer et al., 1984; Lin et al., 1985; Padgett et al., 1984; Ruskin et al., 1984). A fundamental question in the splicing field is how the 5' and 3' splice sites are recognized and paired during the splicing reaction. Recent work in the splicing field has established that a network of RNA interactions may form the structural foundation of the spliceosomes. Possible solutions to many unsolved puzzles are getting attention. RNA-RNA interactions now appear to underlie many aspects of substrate recognition, reaction partner juxtaposition and catalysis. In this article we have presented the latest mechanisms involved in the pre-mRNA splicing and their implication in applied research including cancer.
RNA molecules commonly consist of helical regions separated by internal loops, and in many cases these internal loops have been found to assume stable structures. We have examined the function and dynamics of an internal loop, J5/5a, that joins the two halves of the P4-P6 domain of the Tetrahymena self-splicing group I intron. P4-P6 RNAs with mutations in the J5/5a region showed nondenaturing gel electrophoretic mobilities and levels of Fe(II)-EDTA cleavage protection intermediate between those of wild-type RNA and a mutant incapable of folding into the native P4-P6 tertiary structure. Mutants with the least structured J5/5a loops behaved the most like wild-type P4-P6, and required smaller amounts of Mg2+ to rescue folding. The activity of reconstituted introns containing mutant P4-P6 RNAs correlated similarly with the nature of the J5/5a mutation. Our results suggest that, in solution, the P4-P6 RNA is in a two-state equilibrium between folded and unfolded states. We conclude that this internal loop mainly acts as a flexible hinge, allowing the coaxially stacked helical regions on either side of it to interact via specific tertiary contacts. To a lesser extent, the specific bases within the loop contribute to folding. Furthermore, it is crucial that the junction remain unstructured in the unfolded state. These conclusions cannot be derived from a simple examination of the P4-P6 crystal structure (Cate JH et al., 1996, Science 273:1678-1685), showing once again that structure determination must be supplemented with mutational and thermodynamic analysis to provide a complete picture of a folded macromolecule.
Splicing of nuclear mRNA precursors (pre-mRNAs) takes place in the spliceosome, a large and complex ribonucleoprotein. Nuclear pre-mRNA splicing and group II intron self-splicing occur by a chemically identical pathway involving recognition of a specific branchpoint adenosine and nucleophilic activation of its 2'-hydroxyl group. The chemical similarity between these two splicing reactions, as well as other considerations, have suggested that the catalytic core of the spliceosome and group II introns may be related. Here we test this hypothesis by analyzing splicing and RNA branch formation of a pre-mRNA and a group II intron in which the branchpoint adenosine was substituted with purine base analogues. We find that replacement of the branchpoint adenosine with either of two modified adenosine analogues or guanosine leads to remarkably similar patterns of splicing and RNA branch formation in the two systems.