Essential nucleotide sequences and secondary structure elements of the hairpin ribozyme.

In vitro selection experiments have been used to isolate active variants of the 50 nt hairpin catalytic RNA motif following randomization of individual ribozyme domains and intensive mutagenesis of the ribozyme-substrate complex. Active and inactive variants were characterized by sequencing, analysis of RNA cleavage activity in cis and in trans, and by substrate binding studies. Results precisely define base-pairing requirements for ribozyme helices 3 and 4, and identify eight essential nucleotides (G8, A9, A10, G21, A22, A23, A24 and C25) within the catalytic core of the ribozyme. Activity and substrate binding assays show that point mutations at these eight sites eliminate cleavage activity but do not significantly decrease substrate binding, demonstrating that these bases contribute to catalytic function. The mutation U39C has been isolated from different selection experiments as a second-site suppressor of the down mutants G21U and A43G. Assays of the U39C mutation in the wild-type ribozyme and in a variety of mutant backgrounds show that this variant is a general up mutation. Results from selection experiments involving populations totaling more than 10(10) variants are summarized, and consensus sequences including 16 essential nucleotides and a secondary structure model of four short helices, encompassing 18 bp for the ribozyme-substrate complex are derived.     

A conserved base pair within helix P4 of the Tetrahymena ribozyme helps to form the tertiary structure required for self-splicing.

Site-specific mutagenesis of the self-splicing Tetrahymena intron has been used to investigate the function of C109-G212, a conserved base pair in the P4 stem of group I introns. Mutation of C109 to G affects splicing only slightly, whereas mutation of G212 to A or C reduces the rate of splicing substantially (500-fold reduction in kcat/Km under standard in vitro splicing conditions for the G212C mutant). Splicing activity of the compensatory double mutant (C109G:G212C) is intermediate between those of the two single mutants. Thus, the stability of the P4 stem as well as the identity of the base at position 212 are important for self-splicing. Single and double mutants containing the G212C substitution have a decreased temperature optimum for self-splicing and are partially Mg2+ suppressible, both indicative of structural destabilization. Chemical structure mapping indicates that the mutations do not redirect the global folding of the RNA, but affect the structure locally and at one other site (A183) that is distant in the secondary structure. We propose that, in addition to its pairing in P4, G212 is involved in a base triplet or an alternate base pair that contributes to the catalytically active tertiary structure of the ribozyme.     

RNA splicing: group I intron crystal structures reveal the basis of splice site selection and metal ion catalysis

The group I intron has served as a model for RNA catalysis since its discovery 25 years ago. Four recently determined high-resolution crystal structures complement extensive biochemical studies on this system. Structures of the Azoarcus, Tetrahymena and bacteriophage Twort group I introns mimic different states of the splicing or ribozyme reaction pathway and provide information on splice site selection and metal ion catalysis. The 5'-splice site is selected by formation of a conserved G.U wobble pair between the 5'-exon terminus and the intron. The 3'-splice site is identified through stacking of three base triples, in which the middle triple contains the conserved terminal nucleotide of the intron, OmegaG. The structures support a two-metal-ion mechanism for group I intron splicing that might have corollaries to group II intron and pre-mRNA splicing by the spliceosome.     

截短绿色荧光蛋白突变体反式剪接修复核酶

Ⅰ型内含子核酶经过设计特定的信号引导序列（IGS）,可特异性地定点剪接目的基因RNA,从而在RNA水平达到修复病变基因的目的。以四膜虫材料,克隆了其26S rRNA内含子核酶基因,体外转录证实该I型内含子核酶具有完全的自我剪接的功能。为检测该核酶的反式剪接功能,构建了缺失后半段564bp基因序列的绿色荧光蛋白（GFP）的截短突变体重组质粒XYQ5/XYQ10pEGFP-C-2,并证实其失去了发射绿色荧光的活性。利用PCR和分子克隆技术,构建了以上EGFP突变体的反式剪接修复核酶ptrans-rib-CMV2,该核酶载体以克隆的26S RNA内含子为核心,选择EGFP编码区194位TG为剪接位点,以188-193位设计IGS序列,核酶3′端携带195-890bp的EGFP基因序列,连接于pRC-CMV2真核表达载体中。体外转录突变EGFP的原核表达载体XYQ5/10-pGEM和ptrans-rib-CMV₂,以混合转录产物为模板进行RT-PCR,电泳及测序证实产物中含有反式剪接修复的野生型EGFP mRNA,从而证实构建的反式剪接核酶具有体外反式剪接功能。将截短突变重组质粒XYQ5/XYQ10- pEGFP-C₂与核酶质粒ptrans-rib-CMV₂共转染Hela细胞,用荧光显微镜观察转染结果,发现有少量共转染的Hela细胞发出绿光;RT-PCR检测出野生型EGFP mRNA,证明构建的反式剪接核酶具有体内反式剪接的功能,但其反式剪接效率低。     

Minimum secondary structure requirements for catalytic activity of a self-splicing group I intron

We have completed a comprehensive deletion analysis of the Tetrahymena ribozyme in order to define the minimum secondary structure requirements for phosphoester transfer activity of a self-splicing group I intron. A total of 299 nucleotides were removed in a piecewise fashion, leaving a catalytic core of 114 nucleotides that form 7 base-paired structural elements. Among the various deletion mutants are a 300-nucleotide single-deletion mutant and a 281-nucleotide double-deletion mutant whose activity exceeds that of the wild type when tested under physiologic conditions. Consideration of those structural elements that are essential for catalytic activity leads to a simplified secondary structure model of the catalytic core of a group I intron.     

Modulating the splicing activity of Tetrahymena ribozyme via RNA self-assembly

The internal guiding sequence (IGS) is normally located at the 5' end of trans-splicing ribozymes that are derived from the Tetrahymena group I intron, and is required for the recognition of substrate RNAs and for trans-splicing reactions. Here, we separated the Tetrahymena group I intron at the L2 loop to produce two fragments: the IGS-containing substrate, and the IGS-lacking ribozyme. We show here that two fragments can complex not through the IGS interaction but under the guidance of appended interacting nucleotides, and perform trans-splicing. The splicing reactions took place both in vitro and in mammalian cells, and the spliced mRNA product from the self-assembled ribozyme complex can be translated into functional proteins in vivo. The splicing efficiency was dependent on the length of appending nucleotides.     

Analysis of the P7 region within the catalytic core of the Tetrahymena ribozyme by employing in vitro selection.

The highly conserved P7 region is generally believed to act as a major portion of the catalytic site in the Group I intron ribozyme. However, its functions have not been elucidated except for the fact that it specifically binds a cofactor guanosine required for self-splicing reaction. We attempted an in vitro selection experiment to determine the sequence requirements of this region in the mechanism of catalysis by using the Tetrahymena ribozyme. We found that the selected active clones have the secondary structure similar to that of the wild type with few exceptions. However, their primary sequences were not conserved except G264 and C311 that are the major elements of the binding site for the guanosine. Our results suggest that the unique secondary structure of the P7 region is a primary requisite for the catalytic function of this class of ribozymes.     

Trans-activation of the Tetrahymena group I intron ribozyme via a non-native RNA-RNA interaction

The peripheral P2.1 domain of the Tetrahymena group I intron ribozyme has been shown to be non-essential for splicing. We found, however, that separately prepared P2.1 RNA efficiently accelerates the 3' splice-site-specific hydrolysis reaction of a mutant ribozyme lacking both P2.1 and its upstream region in trans. We report here the unusual properties of this trans-activation. Compensatory mutational analysis revealed that non-native long-range base-pairings between the loop region of P2.1 RNA and L5c region of the mutant ribozyme are needed for the activation in spite of the fact that P2.1 forms base-pairings with P9.1 in the Tetrahymena ribozyme. The trans -activation depends on the non-native RNA-RNA interaction together with the higher order structure of P2.1 RNA. This activation is unique among the known trans-activations that utilize native tertiary interactions or RNA chaperons.     

Template-directed interference footprinting for RNA based on inosine-specific cleavage

We report here the development of a Template-directed Interference (TDI) footprinting assay for RNA. The TDI nucleotide analogue inosine (I) lacks the exocyclic amine of G and is a suitable probe for the role of this group in RNA structure and function. Using an I-specific cleavage protocol we identified three functionally significant G residues in the Tetrahymena ribozyme. These residues are proximal to the active site of the folded intron and likely contribute to the positioning of substrates at the catalytic core.     

Leaving group stabilization by metal ion coordination and hydrogen bond donation is an evolutionarily conserved feature of group I introns.

To understand the behavior of group I introns on a biologically fundamental level, we must distinguish those traits that arise as the products of natural selection (selected traits) from those that arise as the products of neutral drift (non-selected traits). In practice, this distinction relies on comparing the similarities and differences among widely divergent introns to identify conserved traits. Here we address whether the strategies used by the eukaryotic group I intron from the Tetrahymena ciliate to stabilize the leaving group during splicing are maintained in the group I intron from the widely divergent Azoarcus bacterium. A substrate analogue containing a 3'-phosphorothiolate linkage, in which a sulfur atom replaces the bridging 3'-oxygen atom of the scissile phosphate, reacts 20-fold slower in the Azoarcus reaction than the corresponding unmodified substrate in the presence of Mg(II) as the only divalent cation. However, Mn(II) relieves this negative effect such that the 3'-S-P bond cleaves 21-fold faster than does the 3'O-P bond. Other thiophilic divalent metal ions such as Co(II), Cd(II), and Zn(II) similarly support cleavage of the S-P bond. These results indicate that a metal ion directly coordinates to the leaving group in the transition state of the Azoarcus ribozyme reaction. Additionally, the 3'-sulfur substitution eliminates the approximately 10(3)-fold contribution of the adjacent 2'-OH to transition state stabilization. Considering that sulfur accepts hydrogen bonds weakly compared to oxygen, this result suggests that the 2'-OH contributes to catalysis by donating a hydrogen bond to the 3'-oxygen leaving group in the transition state, presumably acting in conjunction with the metal ion to stabilize the developing negative charge. These same catalytic strategies of metal ion coordination and hydrogen bond donation operate in the Tetrahymena ribozyme reaction, suggesting that these features of catalysis have been conserved during evolution and thus extend to all group I introns. The two ribozymes also exhibit quantitative differences in their response to 3'-sulfur substitution. The Azoarcus ribozyme binds and cleaves the phosphorothiolate substrate more efficiently relative to the natural substrate than the Tetrahymena ribozyme under the same conditions, suggesting that the Azoarcus ribozyme better accommodates the phosphorothiolate at the active site both in the ground state and in the transition state. These differences may reflect either a less tightly knit Azoarcus structure and/or spatial deviations between backbone atoms in the two ribozymes that arise during divergent evolution, analogous to the well-documented relationship between protein sequence and structure.     

Probing the mechanisms of DEAD-box proteins as general RNA chaperones: the C-terminal domain of CYT-19 mediates general recognition of RNA

The DEAD-box protein CYT-19 functions in the folding of several group I introns in vivo and a diverse set of group I and group II RNAs in vitro. Recent work using the Tetrahymena group I ribozyme demonstrated that CYT-19 possesses a second RNA-binding site, distinct from the unwinding active site, which enhances unwinding activity by binding nonspecifically to the adjacent RNA structure. Here, we probe the region of CYT-19 responsible for that binding by constructing a C-terminal truncation variant that lacks 49 amino acids and terminates at a domain boundary, as defined by limited proteolysis. This truncated protein unwinds a six-base-pair duplex, formed between the oligonucleotide substrate of the Tetrahymena ribozyme and an oligonucleotide corresponding to the internal guide sequence of the ribozyme, with near-wild-type efficiency. However, the truncated protein is activated much less than the wild-type protein when the duplex is covalently linked to the ribozyme or single-stranded or double-stranded extensions. Thus, the active site for RNA unwinding remains functional in the truncated CYT-19, but the site that binds the adjacent RNA structure has been compromised. Equilibrium binding experiments confirmed that the truncated protein binds RNA less tightly than the wild-type protein. RNA binding by the compromised site is important for chaperone activity, because the truncated protein is less active in facilitating the folding of a group I intron that requires CYT-19 in vivo. The deleted region contains arginine-rich sequences, as found in other RNA-binding proteins, and may function by tethering CYT-19 to structured RNAs, so that it can efficiently disrupt exposed, non-native structural elements, allowing them to refold. Many other DExD/H-box proteins also contain arginine-rich ancillary domains, and some of these domains may function similarly as nonspecific RNA-binding elements that enhance general RNA chaperone activity.     

An in vivo selection method to optimize trans-splicing ribozymes

Group I intron ribozymes can repair mutated mRNAs by replacing the 3′-terminal portion of the mRNA with their own 3′-exon. This trans-splicing reaction has the potential to treat genetic disorders and to selectively kill cancer cells or virus-infected cells. However, these ribozymes have not yet been used in therapy, partially due to a low in vivo trans-splicing efficiency. Previous strategies to improve the trans-splicing efficiencies focused on designing and testing individual ribozyme constructs. Here we describe a method that selects the most efficient ribozymes from millions of ribozyme variants. This method uses an in vivo rescue assay where the mRNA of an inactivated antibiotic resistance gene is repaired by trans-splicing group I intron ribozymes. Bacterial cells that express efficient trans-splicing ribozymes are able to grow on medium containing the antibiotic chloramphenicol. We randomized a 5′-terminal sequence of the Tetrahymena thermophila group I intron and screened a library with 9 × 10⁶ ribozyme variants for the best trans-splicing activity. The resulting ribozymes showed increased trans-splicing efficiency and help the design of efficient trans-splicing ribozymes for different sequence contexts. This in vivo selection method can now be used to optimize any sequence in trans-splicing ribozymes.     

In vitro selection of second site revertants analysis of the hairpin ribozyme active site

We have used in vitro genetics to evaluate the function and interactions of the conserved base G8 in the hairpin ribozyme catalytic RNA. Second site revertant selection for a G8X mutant, where X is any of the other three natural nucleobases, yielded a family of second site suppressors of the G8U mutant, but not of G8C or G8A, indicating that only G and U can be tolerated at position 8 of the ribozyme. This result is consistent with recent observations that point to the functional importance of G8 N-1 in the chemistry of catalysis by this ribozyme reaction. Suppression of the G8U mutation was observed when changes were made directly across loop A from the mutated base at substrate position +2 or positions +2 and +3 in combination. The same changes made in the context of the natural G8 sequence resulted in a very large drop in activity. Thus, the G8U mutation results in a change in specificity of the ribozyme from 5'-N / GUC-3' to 5'-N / GCU-3'. The results presented imply that G8 interacts directly with U+2 during catalysis. We propose that this interaction favors the correct positioning of the catalytic determinants of G8. The implications for the folding of the ribozyme and the catalytic mechanism are discussed.     

Mutations at the guanosine-binding site of the Tetrahymena ribozyme also affect site-specific hydrolysis.

Self-splicing group I introns use guanosine as a nucleophile to cleave the 5' splice site. The guanosine-binding site has been localized to the G264-C311 base pair of the Tetrahymena intron on the basis of analysis of mutations that change the specificity of the nucleophile from G (guanosine) to 2AP (2-aminopurine ribonucleoside) (F. Michel et al. (1989) Nature 342, 391-395). We studied the effect of these mutations (G-U, A-C and A-U replacing G264-C311) in the L-21 ScaI version of the Tetrahymena ribozyme. In this enzymatic system (kcat/Km)G monitors the cleavage step. This kinetic parameter decreased by at least 5 x 10(3) when the G264-C311 base pair was mutated to an A-U pair, while (kcat/Km)2AP increased at least 40-fold. This amounted to an overall switch in specificity of at least 2 x 10(5). The nucleophile specificity (G > 2AP for the G-C and G-U pairs, 2AP > G for the A-U and A-C pairs) was consistent with the proposed hydrogen bond between the nucleotide at position 264 and N1 of the nucleophile. Unexpectedly, the A-U and A-C mutants showed a decrease of an order of magnitude in the rate of ribozyme-catalyzed hydrolysis of RNA, in which H2O or OH- replaces G as the nucleophile, whereas the G-U mutant showed a decrease of only 2-fold. The low hydrolysis rates were not restored by raising the Mg2+ concentration or lowering the temperature. In addition, the mutant ribozymes exhibited a pattern of cleavage by Fe(II)-EDTA indistinguishable from that of the wild type, and the [Mg2+]1/2 for folding of the A-U mutant ribozyme was the same as that of the wild type. Therefore the guanosine-binding site mutations do not appear to have a major effect on RNA folding or stability. Because changing G264 affects the hydrolysis reaction without perturbing the global folding of the RNA, we conclude that the catalytic role of this conserved nucleotide is not limited to guanosine binding.     

Optimizing the substrate specificity of a group I intron ribozyme

Group I ribozymes can repair mutant RNAs via trans-splicing. Unfortunately, substrate specificity is quite low for the trans-splicing reaction catalyzed by the group I ribozyme from Tetrahymenathermophila. We have used a systematic approach based on biochemical knowledge of the function of the Tetrahymena ribozyme to optimize its ability to discriminate against nonspecific substrates in vitro. Ribozyme derivatives that combine a mutation which indirectly slows down the rate of the chemical cleavage step by weakening guanosine binding with additional mutations that weaken substrate binding have greatly enhanced specificity with short oligonucleotide substrates and an mRNA fragment derived from the p53 gene. Moreover, compared to the wild-type ribozyme, reaction of a more specific ribozyme with targeted substrates is much less sensitive to the presence of nonspecific RNA competitors. These results demonstrate how a detailed understanding of the biochemistry of a catalytic RNA can facilitate the design of customized ribozymes with improved properties for therapeutic applications.     

Accommodation of Ca(II) ions for catalytic activity by a group I ribozyme

The wildtype Tetrahymena ribozyme cannot catalyze detectable levels of phosphotransfer activity in vitro on an exogenous RNA substrate oligonucleotide when calcium(II) is supplied as the only available divalent ion. Nevertheless, low-error mutants of this ribozyme have been acquired through directed evolution that do have activity in 10 mM CaCl₂. The mechanisms for such Ca(II) accommodation are not known. Here, we assayed the entire molecule in an effort to identify the roles of the mutations in allowing catalytic activity in Ca(II). We used four biochemical probing techniques - native-gel electrophoresis, hydroxyl radical footprinting, terbium(III) cleavage footprinting, and phosphorothioate interference mapping - to compare the solution structure of the wildtype ribozyme with that of a Ca(II)-active five-site mutant. We compared the gross folding patterns and specific metal-binding sites in both MgCl₂ and CaCl₂ solutions. We detected no large-scale folding differences between the two RNAs in either metal. However, we did discover a limited number of local folding differences, involving regions of the RNA affected by positions 42, 188, and 270. These data support the notion that Ca(II) is accommodated by the Tetrahymena ribozyme by a slight breathing at the active site, but that alterations at, near to, and distal from the active site can all contribute to Ca(II)-based activity.