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. 2003 Mar 1;31(5):1488–1496. doi: 10.1093/nar/gkg225

Putative intermediary stages for the molecular evolution from a ribozyme to a catalytic RNP

Yoshiya Ikawa 1, Kentaro Tsuda 1, Shigeyoshi Matsumura 1, Shota Atsumi 1, Tan Inoue 1,a
PMCID: PMC149818  PMID: 12595557

Abstract

A hypothetical evolutionary pathway from a ribozyme to a catalytic RNA–protein complex (RNP) is proposed and examined. In this hypothesis for an early phase of molecular evolution, one RNA–RNA interaction in the starting ribozyme is replaced with an RNA–protein interaction via two intermediary stages. At each stage, the original RNA–RNA interaction and a newly introduced RNA–protein interaction are designed to coexist. The catalytic RNPs corresponding to the intermediary stages were constructed by employing the Tetrahymena ribozyme together with molecular modeling. Analyses of the RNPs indicate that the protein can fully replace the original role of the RNA–RNA interaction in the starting ribozyme and that the association of a protein with a ribozyme might be beneficial for improving the ribozymatic activity.

INTRODUCTION

RNA molecules play a variety of roles in cellular biological processes. It has been proposed that they played a major role in the early stage of the evolution of life because they can act as carriers of genetic information in addition to catalytic molecules called ribozymes (1). The stage is called ‘RNA world’, where RNA molecules replicate depending on their catalytic activity. The ‘RNA world’ has been suggested to have evolved into the ‘RNP (RNA–protein) world’ which links the ‘RNA world’ and the modern ‘DNA world’ (2–5).

The early phase of the transition from ‘RNA world’ to ‘RNP world’ presumably depended on gradual replacement of RNA structural elements with protein elements. Recent analyses of ribozymes and RNA–protein complexes (RNPs) support this hypothesis.

Evidence was obtained from the research on group I self-splicing ribozymes (6). In the Tetrahymena ribozyme, its large peripheral domain called P5abc is important for performing efficient splicing reactions under physiological conditions, and its deletion causes considerably reduced activity (7). However, mutant RNA, lacking the domain, can be activated by adding Neurospora CYT-18 protein in trans (6). This demonstrates that CYT-18, which binds to the conserved P4–P6–P6a regions of various group I ribozymes, can functionally replace the role of P5abc.

Another piece of evidence came from structural and functional comparisons of the mammalian mithochondrial ribosome with that of Escherichia coli. The two ribosomes have approximately the same molecular weight, but the ratios of RNA to protein of the mitochondrial and E.coli ribosomes are 1:2 and 2:1, respectively, suggesting that certain RNA components in the E.coli ribosome were replaced by proteins in the mitochondrial ribosome (8). This is supported by comparison between the analysis of the 3-dimensional structure of bacterial ribosomes and the proteomic analysis of the mitochondrial ribosome (9,10). The comparison revealed that the shortened or lost regions of rRNAs in the mitochondrial ribosome were compensated structurally by the protein factors.

In the later phase of the transition, the primordial RNP complexes could have improved their catalytic activity and expanded their functional repertoires, which cannot be achieved by RNA alone. One example can be comprehended in a bacterial RNase P, which processes 5′ leader sequences of precursor tRNAs and consists of a large RNA and a small protein component (11). It has been shown that the RNA component is responsible for tRNA recognition and catalysis so that accurate tRNA processing can proceed without the protein component (12). In this case, the protein component has two roles. First, it dramatically improves turnover efficiency by neutralizing negative charges of the catalytic RNA to improve binding of substrate RNA (13). Second, the repertoire of substrate RNAs is expanded in the presence of the protein component because it recognizes the 5′ leader sequence of substrate RNAs (14–18).

Recently we successfully replaced an RNA–RNA interaction of the Tetrahymena ribozyme with an RNA–protein interaction without sacrificing its activity (19). In the designed RNP, a hardly active RNA component without protein regained its activity when a designed protein possessing two RNA-binding motifs for the RNA’s two regions were supplied in trans. This mimics the molecular evolution from a ribozyme to an RNP (19). However, it still lacks indispensable intermediary stages for fully understanding the evolutionary process because it is hard to imagine that the starting RNA (Fig. 1A) was directly converted to RNP (Fig. 1D) coincidentally at the same time.

Figure 1.

Figure 1

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Abbreviated stages for replacing an intramolecular RNA–RNA interaction in a ribozyme with two RNA–protein interactions. (A) A specific RNA–RNA interaction to be replaced. (B and C) An RNA-binding protein interacts with one or two sites, respectively, in the RNA in the presence of the intramolecular interaction. (D) The RNA–RNA interaction is completely replaced with the RNA–protein interaction and the resulting RNP is inactive without a protein cofactor.

In a process of molecular evolution, significant loss or reduction of the functions of the original RNAs must have been fatal. Thus, it seems plausible that the intermediary stages where functional properties and/or activities of the prototype RNA molecule(s) were maintained could have existed before emergence of the final RNP form. Following this line of thought, we designed a model pathway of artificial evolution from a ribozyme to an RNP by employing the Tetrahymena group I ribozyme as a model RNA molecule.

As a model pathway of artificial evolution of the Tetrahymena ribozyme to a catalytic RNP, we proposed the four model stages, being the ‘initial RNA’ stage, ‘intermediate I’, ‘intermediate II’ and the ‘final RNP’ stage, described as follows. In the ‘intermediate I’ stage, a protein binds to the RNA but hardly plays any role (Fig. 1B). It begins to play a significant role in the ‘intermediate II’ stage by assisting the RNA–RNA interaction in the ribozyme (Fig. 1C). From the ‘intermediate II’ to the ‘final’ stage, the priorities of the RNA–RNA and RNA–protein interactions for the ribozyme are reversed so that the original RNA–RNA interaction can be abolished (Fig. 1D). The resulting complex acts as an ‘RNA–protein complex (RNP)’ because its activity depends on the protein. To assess this model pathway, we constructed and characterized those intermediary molecules.

MATERIALS AND METHODS

Molecular modeling

Protein and RNA molecular modeling was performed by using Insight II on a Silicon Graphics workstation. A molecular model of boxB-11nt/RRE was constructed from the coordinates of the crystal structure of the P4–P6 domain of the Tetrahymena ribozyme [Protein Data Bank ID 1GID (20)], the NMR structure of the bacteriophage λN peptide–boxB RNA complex [1QFQ (21)] and HIV Rev-peptide–RRE RNA complex [1ETF (22)].

Plasmids

Plasmids encoding the Tetrahymena ribozyme (pTZIVSU) and pep A and pep G proteins (pTYBpepA and pTYBpepG) and their derivatives were described previously (19,23). Plasmids encoding newly designed derivatives of the Tetrahymena ribozyme were constructed from pTZIVSU by site-directed mutagenesis as described (24).

Preparation of precursor RNAs and activator proteins

The precursor RNAs and proteins were prepared as described (19). All RNAs employed in this study were prepared by in vitro transcription with T7 RNA polymerase and [α-32P]GTP (25). All proteins for in vitro self-splicing assays were synthesized from plasmid pTYBpepA, pTYBpepG or their derivatives in E.coli strain ER2566, followed by purification with the IMPACT™ T7 System (New England Biolabs) (26).

In vitro splicing assays

In vitro splicing assays were performed as described with some modifications (19). Precursor RNAs (10 nM) labeled with 32P and dissolved in distilled water were incubated at 80°C for 5 min. After cooling and incubation at 37°C for 1 min, the 10-fold concentrated reaction buffer and the protein in the dilution buffer (20 mM Tris–HCl, pH 7.5, 40 mM KCl, 50% glycerol) were simultaneously added. After preincubation for 5 min, the reactions were started by adding 200 µM GTP at 37°C in the presence of 40 mM Tris–HCl, pH 7.5, 1.8 mM MgCl2, 80 mM KCl and 2.5% glycerol. Aliquots were removed at specified times. The reactions were terminated by adding an equal volume of stop solution (150 mM EDTA, 70% formamide, 0.25% xylene cyanol), followed by electrophoresis on 5% polyacrylamide denaturing gels. The RNAs were quantified with a BioImaging Analyzer (BAS2500; Fuji Film, Japan). All experiments were performed in triplicate and the extent of reactions indicated in figures are their average values.

RNase V1 digestion and primer extension analysis

For RNase V1 digestion, L-21 ScaI form RNAs lacking the 5′ and 3′ exons were employed in place of the corresponding precursor RNAs. We dissolved 1.6 pmol of L-21 ScaI RNAs in 34 µl of distilled water and heated the solution at 80°C for 5 min. After cooling and incubation at 37°C for 1 min, 4 µl of the 10-fold concentrated reaction buffer and 2 µl of the protein (final concentration 4 µM) were simultaneously added. After preincubation for 5 min, RNA–protein complexes were digested by adding 2 µl of RNase V1 (0.4 U/ml; Amersham Pharmacia Biotech), followed by incubation for 10 min. The RNAs were extracted with phenol and recovered by ethanol precipitation. RNase V1 cleavage sites were detected by primer extension using two oligonucleotide primers, complementary to positions 201–221 and 298–317 of the wild-type ribozyme. ReverTra Ace (Toyobo, Japan) reverse transcribed 2 pmol of the modified RNAs. Products were separated on 6% polyacrylamide denaturing gels and quantified with a BioImaging Analyzer (BAS 2500; Fuji Film, Japan).

RESULTS AND DISCUSSION

‘Intermediate I’

To design model molecules in our hypothetical evolutionary path from an RNA to an RNP, we attempted to replace an RNA–RNA interaction between the P5b and P6a/b regions of the Tetrahymena self-splicing intron RNA with an RNA–protein–RNA bridge interaction. The RNA–RNA interaction between P5b and P6a/b has been shown to be necessary for establishing the active ribozyme structure (Fig. 2A) (20,27).

Figure 2.

Figure 2

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Molecular designs of catalytic RNPs representing model steps from ribozyme to RNP. (A) Schematic presentation of the secondary structure of the Tetrahymena ribozyme and its derivatives. Original sequences of P5b and P6a/b regions are included. (B) Amino acid sequences of the RNA-binding proteins, pepA and pepG, and their derivatives. Modified amino acids in the RNA-binding domains of mN and mRev are marked with asterisks.

First, a model representing the ‘intermediate I’ (Fig. 1B) was designed in which RNA-binding proteins do not assist the formation of active ribozyme. The P5b region of the ribozyme was modified to bind to the previously designed RNA-binding proteins (pepA and pepG) (19). They share the same modular organization, consisting of an N-terminal region containing one RNA-binding motif [N-peptide motif (21,28)], a linker region and a C-terminal region containing a second RNA-binding motif [Rev-peptide motif (22)] (Fig. 2B). The P5b stem–loop region possessing a GAAA tetraloop was replaced with a boxB stem–loop (boxB) motif possessing a GAAAA pentaloop without changing the length of the P5b stem (Fig. 2A) (29). The boxB motif and its GAAAA pentaloop region have been shown to interact with the N-peptide motif (21,30) and the 11 nucleotide receptor (11ntR) motif (31,32), respectively. Comparison between the NMR structure of the boxB–N-peptide complex and that of the GAAA loop–11ntR complex suggests that the opposite side of the boxB motif can be used for interacting with either N-peptide or 11ntR (21). The modified P5b should, therefore, associate with the N-peptide region of pepA or pepG while retaining RNA– RNA interaction with the 11ntR motif in P6a/b.

Under the assay conditions we employed (1.8 mM MgCl2 and 80 mM KCl, 37°C), the designed RNA termed boxB-11nt RNA performed a self-splicing reaction (Fig. 3A). Its reaction efficacy was somewhat less than that of the wild-type ribozyme probably because the GAAAA pentaloop binds to 11ntR less tightly than the GAAA tetraloop (21,33,34). The activity of the boxB-11nt RNA was not affected by pepA or pepG, which indicates that the protein factors do not influence the ribozyme activity (Fig. 3B). A variant RNA containing a UUCG loop at P5b was virtually inactive, indicating that the P5b–P6a/b interaction is important for establishing the active form under the conditions (Fig. 3B).

Figure 3.

Figure 3

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In vitro self-splicing reactions of RNAs (10 nM) corresponding to ‘intermediate I’ with or without 1 µM pepA or pepG. (A) Time-courses of the splicing reaction with the Tetrahymena ribozyme and boxB-11nt variant representing the ‘intermediate I’. (B) An autoradiogram for the splicing reaction of the 32P-labeled precursor RNAs.

‘Intermediate II’

Next, a model RNA representing the ‘intermediate II’ stage (Fig. 1C) was designed. In this case, both a protein factor and the RNA–RNA interaction can assist the formation of active ribozymes. The second protein-binding motif [RRE (22)] was introduced in the P6b region of boxB-11nt RNA by employing a molecular modeling program to establish the interaction with the Rev motif in pepA or pepG (Fig. 2A). As shown in the model (Fig. 4), the extended structure of the protein bridges P5b and P6b without disrupting the P4–P6 three-dimensional structure.

Figure 4.

Figure 4

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The secondary structure (left) and three-dimensional model (right) of the P4–P6 domain of the catalytic RNP represent the ‘intermediate II’. For clarity, a view from the reverse side of the structure in the left panel is depicted on the right-hand side.

The resulting RNA molecule termed boxB-11nt/RRE RNA was subjected to the splicing reaction with or with out the protein (Fig. 5A). In the absence of any protein, boxB-11nt/RRE RNA was still active (the final extent of the reaction was 49%; the relative amount of the precursor RNA spliced at the final time point), indicating that RNA–RNA interaction is retained. However, its activity was slightly weaker than boxB-11nt RNA (59%), presumably because the RRE being located close to 11ntR somewhat distorts the structure of 11ntR (Figs 4 and 5). The splicing of boxB-11nt/RRE RNA was enhanced with pepG or pepA (Fig. 5), whereas boxB-11nt RNA and the wild-type were inert to the proteins (Fig. 3B). These data suggest that both the protein and RNA–RNA interactions contribute to activating boxB-11nt/RRE RNA. Interestingly, the designed RNP exhibited activity comparable to (in the case of a complex with pepG, 72%) or higher than (in the case of a complex with pepA, 83%) that of the parental Tetrahymena ribozyme (70%) or the ‘intermediate I’ (boxB-11nt, 59%). This demonstrates that formation of the RNP is advantageous for RNA catalysis in this case.

Figure 5.

Figure 5

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In vitro self-splicing reaction of designed RNAs representing ‘intermediate II’. (A) An autoradiogram of 32P-labeled precursor RNAs: mN and mRev correspond to pepA-mN and pepA-mRev, respectively; in vitro self-splicing reaction of boxB-11nt/RRE and its RRE mutant (10 nM), with or without 1 µM protein. (B) Time-courses of self-splicing reactions with boxB-11nt/RRE, with and without the proteins, pepA or pepG. (C) An autoradiogram of the in vitro self-splicing reaction with 32P-labeled precursor RNAs; the reaction of boxB-11nt/RRE and its derivatives with the original P5b or a UUCG replacing the GAAAA loop, with or without 1 µM protein.

To see whether the activation of the ribozyme depends on the RNA–peptide interaction, variant RNAs of ‘intermediate II’ and proteins were designed and investigated (Fig. 2). boxB-11nt/mutRRE RNA, possessing a mutated RRE, was designed to attenuate the REV–RRE interaction. When compared with boxB-11nt/RRE RNA, this RNA retained the activity without protein (43–49%) but reduced the final extent of the reaction from 72 to 50% and 83 to 55% in the presence of pepG and pepA, respectively, when compared with boxB-11nt/RRE RNA (Fig. 5). Two pepA protein variants, pepA-mN and pepA-mRev (Fig. 2B), whose N-motif and Rev motif were mutated to weaken the RNA-binding ability, did not activate and very weakly activated the RNA, respectively (Fig. 5A). As expected, these indicate that the activation of boxB-11nt/RRE RNA by the protein depends on two RNA–peptide interactions and that the RNA–RNA interaction between P5b and P6a/b contributes to the basal activity of the ‘intermediate II’ molecule in the absence of protein.

boxB-11nt/RRE RNA, whose P5b boxB was replaced by the original Tetrahymena P5b sequence (GAAA-11nt/RRE), was also prepared (Fig. 2A). The activity of the GAAA-11nt/RRE RNA (61%) was slightly higher than that of the boxB-11nt/RRE RNA (49%), presumably because the GAAA tetraloop can bind to 11ntR more tightly than the GAAAA pentaloop (21,31,32). Interestingly, this variant was activated considerably by the protein, suggesting that the Tetrahymena P5b region can interact with N-peptide (Fig. 5C). This is consistent with previous observations, where (i) N-peptide interacts with a variant boxB sequence possessing a GAAA tetraloop (21) and (ii) the phosphate backbone is the major element responsible for the interaction between the N-peptide and the stem region of boxB (21,30).

The protein-dependent ribozyme

To design the molecule corresponding to the ‘final RNP’ stage (Fig. 1D), the P5b–P6a/b RNA–RNA interaction was eliminated from boxB-11nt/RRE RNA by mutating its 11ntR motif in the P6a/b region (Fig. 2A). Because the P5b–P6a/b RNA–RNA interaction is critical for establishing an active three-dimensional structure of the Tetrahymena ribozyme (20,27), the resulting boxB-mut11ntR/RRE RNA displayed very weak activity (4%) in the absence of the protein (Fig. 6). However, in the presence of the protein, an efficient splicing reaction was observed (57% with pepG and 72% with pepA), comparable with that by the parental Tetrahymena ribozyme (70%) (Fig. 6). boxB-mut11ntR/RRE RNA did not react with pepA-mN or pepA-mRev, indicating that the activation depends on two RNA-binding regions in the protein, as is the case for boxB-11nt/RRE RNA. These data suggest that the complete replacement of the RNA–RNA interaction with the RNA–protein–RNA bridge interaction is acceptable for the ribozyme.

Figure 6.

Figure 6

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In vitro self-splicing reaction of protein-dependent ribozymes. (A) An autoradiogram of the reaction with 32P-labeled precursor RNAs is shown: mN and mRev correspond to pepA-mN and pepA-mRev, respectively; in vitro self-splicing reaction of boxB-mut11nt/RRE (10 nM), with or without 1 µM protein. (B) Time-courses of the splicing reaction with the boxB-mut11nt/RRE in the presence or absence of an activator protein.

RNA–protein interactions in the ‘intermediate II’ stage

To see whether the designed RNA–protein interactions exist in the actual construction, we performed an RNase interference analysis with and without pepA by employing RNase V1, because the specifically protected nucleotides in the boxB and RRE motifs in the presence of N-peptide or Rev-peptide are well known (19,33,34). In the case of boxB-11nt/RRE RNA, the previously identified residues at both protein-binding sites were protected by the presence of pepA, indicating that pepA interacts with both the boxB and RRE regions in the RNA (Fig. 7A). Chemical modification was also attempted using dimethyl sulfate (DMS). The adenine of the 11ntR region (position 225) is known to be protected from modification when the P5b–P6a/b RNA–RNA interaction is present (35). It is similarly protected by boxB-11nt/RRE, with or without pepA (data not shown). Although these experiments only provide information on an ensemble of molecules, the data are consistent with the 3-dimensional model for the ‘intermediate II’ (Fig. 4), where RNA–RNA and RNA–protein interactions coexist.

Figure 7.

Figure 7

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(A) RNA footprints with RNase V1 in the presence of pepA or its mutants. Autoradiograms are shown for RNase V1 footprints of the boxB (upper) and RRE (lower) regions of boxB-11nt/RRE. Dots indicate sites that show protection from RNase V1. (B) Degree of RNase V1 cleavage on the boxB or RRE region of boxB-11nr/RRE in the presence of pepA or its mutants. (C) Degree of RNase V1 cleavage on the boxB or RRE region of boxB-11nr/RRE and its mutants in the presence or absence of pepA.

In the presence of pepA-mN, which has mutations in the N-peptide motif, no protection from RNase V1 was observed for the boxB motif (Fig. 7B, left). In the presence of pepA-mRev, which has mutations in the Rev-peptide motif, no protection was observed for RRE (Fig. 7B, right). However, the boxB and RRE were weakly protected in the presence of pepA-mRev and pepA-mN, respectively (Fig. 7B). The boxB region of boxB-11nt/RRE RNA was protected to a greater extent than its mutRRE variant in the presence of pepA (Fig. 7C). These data support the model that the two RNA–peptide interactions act cooperatively or that the formation of one RNA–peptide interaction facilitates the formation of another.

Concentration dependence of the protein-assisted splicing reaction

Bimolecular complexes containing two binding units in each component are classified as ‘pseudoallosteric’ if the two units act cooperatively (36). Its binding profile exhibits a sigmoidal curve and its Hill coefficient is much greater than 1 (36). However, if not pseudoallosteric, the binding profile exhibits a Michaelis–Menten type curve and its Hill coefficient is 1.

To further characterize two RNA–peptide binding units in the RNA–protein complex, the relationship between protein concentration and splicing activity was investigated by using boxB-11nt/RRE RNA and boxB-mut11ntR/RRE RNA. A sigmoidal curve depending on the protein concentration was observed for the two RNAs (Fig. 8A). The Hill coefficients of boxB-11nt/RRE and boxB-mut11ntR/RRE RNA for pepA were 2.4 and 2.5, respectively, whereas those for pepG were 3.9 and 4.0, respectively. Thus these data indicate that two RNA–peptide binding sites act cooperatively.

Figure 8.

Figure 8

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The splicing reactions of boxB-11nt/RRE and boxB-mut11nt/RRE (10 nM) were tested in the presence of various concentrations of pepA or pepG. The y-axis was plotted as raw data (extent of the splicing reaction) (A) or normalized values (B). At each protein concentration, splicing reactions were performed three times. The results were identical so that the error bars were omitted for the clarity of the figures.

The dissociation constants (Kd values) of the RNP, consisting of boxB-11nt/RRE or boxB-mut11ntR/RRE RNA with the protein, deduced by Hill plots were very close, despite the fact that their activity levels without the protein were quite different. The Kd of boxB-11nt/RRE with pep A or pepG was 450 or 790 nM, whereas that of boxB-mut11nt/RRE with pepA or pepG was 440 or 790 nM. Moreover, if their curves were re-plotted by normalizing the basal activity as Y = 0 and the maximal activity as Y = 1 (Fig. 8B), the normalized curves largely overlapped except at one point, as follows. As an exception, a weak cooperativity between the RNA–RNA and RNA–protein interaction seems observable in the pepA and boxB-11nt/RRE combination because the boxB-11nt/RRE, but not boxB-mut11ntR/RRE RNA, was activated in the presence of 250 nM pepA.

The affinity of pepA and pepG for the RNAs was essentially independent of the RNA–RNA interaction between P5b– P6a/b because boxB-11nt/RRE and boxB-mut11nt/RRE showed similar affinity for the activator proteins. In other words, the RNA–protein–RNA bridge forms independently of the intramolecular RNA–RNA interaction. Thus, for a single boxB-11ntR/RRE RNA molecule, it seems to be destined to produce an active RNP if the RNA–RNA or RNA–protein interaction is formed. Consequently, the activity of the whole reaction mixture is likely enhanced, depending on the number of molecules in the mixture that are converted from ones without the interaction to those with either one or both of the RNA–RNA and RNA–protein interactions. Thus the major folding process of the boxB-11nt/RRE RNA in the presence of the protein could be drawn as two branched pathways in which the RNA molecules utilize either the RNA–RNA or RNA–protein interaction as a trigger to establish the active three-dimensional structure.

Comparison with natural ribozymes

In the present study, we examined a hypothetical evolutionary pathway from a ribozyme to a catalytic RNA–protein complex (RNP). The model pathway involves an intermediary stage where functional RNA–RNA and RNA–protein interactions coexist (Fig. 1C). It is infeasible to determine whether, in the early phase of the transition from ‘RNA world’ to ‘RNP world’, the gradual replacement of the RNA elements with protein elements proceeded in a manner similar to the present model. There is, however, a fact perhaps corresponding to an example of such a ‘ribozyme to RNP’ transition (37). Ten group I introns are found in the mitochondrial genome of Neurospora crassa. Four of them lacking the P5abc activator unit depend on the CYT-18 protein factor to conduct their splicing reactions (38–40) whereas the three introns with an intact P5abc perform splicing without CYT-18 (37). These CYT-18-dependent and -independent introns seem to correspond to the last (Fig. 1D) and initial (Fig. 1A) stages of the ‘ribozyme to RNP’ transition, respectively.

Interestingly, among the rest of the introns, two possess a short P5abc retaining the highly conserved element, the A-rich bulge (or P5a), but lacking one (P5b) or two (P5b and P5c) unconserved elements (37). The short forms of P5abc are likely functional because (i) the A-rich bulge is completely identical to the consensus (41) and (ii) mutational analyses using the Tetrahymena and Aspergillus group I intron ribozymes have demonstrated that a short P5abc possessing only the bulge still retains the ability to activate the ribozyme (42,43). In vivo splicing of the introns with the short P5abc is inhibited by mutation of CYT-18 protein (39,44). These facts suggest that they may utilize both the RNA activator unit and the protein factor like the designed ‘intermediate II’ or boxB-11nt/RRE in the present study. This is consistent with the phylogenetic data suggesting that CYT-18 presumably emerged later than P5abc in evolution. Thus it seems worth proposing a model in which the introns with the short P5abc are the transition state from ‘group I ribozyme’ to ‘group I catalytic RNP’ and thus they are equivalent to the ‘intermediate II’ of our model.

Conclusion

A feasible molecular evolutionary pathway from a ribozyme to a catalytic RNP was designed and examined by employing model molecules that represent its intermediary stages. The model molecules were designed based on the high resolution three-dimensional structure of a Tetrahymena ribozyme domain and two RNA–peptide complexes.

A model molecule designed to mimic a catalytic RNP (boxB-mut11ntR/RRE RNA) lacks a functionally important RNA–RNA interaction of the parental Tetrahymena ribozyme. The RNA, which was hardly active by itself, became as active as the parental ribozyme in the presence of an RNA-binding protein (pepA). The result indicates that certain natural RNA–RNA interactions in the ribozyme can be fully replaced by protein–RNA interactions.

Another model molecule designed to mimic the putative intermediary stage where the RNA–RNA and RNA–protein interactions coexist was considerably active without protein and became more active than the parental Tetrahymena ribozyme. This indicates that the association of a ribozyme with a protein might be advantageous for improving the activity of the ribozyme. The RNAs corresponding to the intermediary stage of the present model might exist in the group I introns in Neurospora because a protein factor seems to enhance the splicing reaction of its intron RNAs containing a short P5 RNA which probably functions as a weak activator in cis.

Acknowledgments

ACKNOWLEDGEMENTS

We thank the members of the Inoue Laboratory for critical reading of the manuscript. This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (T.I.) and the Encouragement of Young Scientists (Y.I.) from the Ministry of Education, Science, Sports and Culture, Japan.

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