Trans-splicing of a mutated glycosylasparaginase mRNA sequence by a group I ribozyme deficient in hydrolysis.

RNA reprogramming represents a new concept in correcting genetic defects at the RNA level. However, for the technique to be useful for therapy, the level of reprogramming must be appropriate. To improve the efficiency of group I ribozyme-mediated RNA reprogramming, when using the Tetrahymena ribozyme, regions complementary to the target RNA have previously been extended in length and accessible sites in the target RNAs have been identified. As an alternative to the Tetrahymena model ribozyme, the DiGIR2 group I ribozyme, derived from a mobile group I intron in rDNA of the myxomycete Didymium iridis, represents a new and attractive tool in RNA reprogramming. We reported recently that the deletion of a structural element within the P9 domain of DiGIR2 turns off hydrolysis at the 3' splice site (side reaction) without affecting self-splicing [Haugen, P., Andreassen, M., Birgisdottir, A.B. & Johansen, S.D. (2004) Eur. J. Biochem. 271, 1015-1024]. Here we analyze the potential of the modified ribozyme, deficient in hydrolysis at the 3' splice site, for application in group I ribozyme-mediated trans-splicing of RNA. The improved ribozyme catalyses both cis-splicing and trans-splicing in vitro of a human glycosylasparaginase mRNA sequence with the same efficiency as the original DiGIR2 ribozyme, but without detectable levels of the unwanted hydrolysis.     

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.     

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。     

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.     

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.     

Naegleria nucleolar introns contain two group I ribozymes with different functions in RNA splicing and processing.

We have characterized the structural organization and catalytic properties of the large nucleolar group I introns (NaSSU1) of the different Naegleria species N. jamiesoni, N. andersoni, N. italica, and N. gruberi. NaSSU1 consists of three distinct RNA domains: an open reading frame encoding a homing-type endonuclease, and a small group I ribozyme (NaGIR1) inserted into the P6 loop of a second group I ribozyme (NaGIR2). The two ribozymes have different functions in RNA splicing and processing. NaGIR1 is an unusual self-cleaving group I ribozyme responsible for intron processing at two internal sites (IPS1 and IPS2), both close to the 5' end of the open reading frame. This processing is hypothesized to lead to formation of a messenger RNA for the endonuclease. Structurally, NaGIR2 is a typical group IC1 ribozyme, catalyzing intron excision and exon ligation reactions. NaGIR2 is responsible for circularization of the excised intron, a reaction that generates full-length RNA circles of wild-type intron. Although it is only distantly related in primary sequence, NaSSU1 RNA has a predicted organization and function very similar to that of the mobile group I intron DiSSU1 of Didymium, the only other group I intron known to encode two ribozymes. We propose that these twin-ribozyme introns define a distinct category of group I introns with a conserved structural organization and function.     

Computational prediction of efficient splice sites for trans-splicing ribozymes

Group I introns have been engineered into trans-splicing ribozymes capable of replacing the 3'-terminal portion of an external mRNA with their own 3'-exon. Although this design makes trans-splicing ribozymes potentially useful for therapeutic application, their trans-splicing efficiency is usually too low for medical use. One factor that strongly influences trans-splicing efficiency is the position of the target splice site on the mRNA substrate. Viable splice sites are currently determined using a biochemical trans-tagging assay. Here, we propose a rapid and inexpensive alternative approach to identify efficient splice sites. This approach involves the computation of the binding free energies between ribozyme and mRNA substrate. We found that the computed binding free energies correlate well with the trans-splicing efficiency experimentally determined at 18 different splice sites on the mRNA of chloramphenicol acetyl transferase. In contrast, our results from the trans-tagging assay correlate less well with measured trans-splicing efficiency. The computed free energy components suggest that splice site efficiency depends on the following secondary structure rearrangements: hybridization of the ribozyme's internal guide sequence (IGS) with mRNA substrate (most important), unfolding of substrate proximal to the splice site, and release of the IGS from the 3'-exon (least important). The proposed computational approach can also be extended to fulfill additional design requirements of efficient trans-splicing ribozymes, such as the optimization of 3'-exon and extended guide sequences.     

Identification of the most accessible sites to ribozymes on the hepatitis C virus internal ribosome entry site

The hepatitis C virus (HCV) is a major causative agent of chronic hepatitis and hepatocellular carcinoma. The development of alternative antiviral therapies is warranted because current treatments for the HCV infection affect only a limited number of patients and lead to significant toxicities. The HCV genome is exclusively present in the RNA form; therefore, ribozyme strategies to target certain HCV sequences have been proposed as anti-HCV treatments. In this study, we determined which regions of the internal ribosome entry site (IRES) of HCV are accessible to ribozymes by employing an RNA mapping strategy that is based on a trans-splicing ribozyme library. We then discovered that the loop regions of the domain IIIb of HCV IRES appeared to be particularly accessible. Moreover, to verify if the target sites that were predicted to be accessible are truly the most accessible, we assessed the ribozyme activities by comparing not only the trans-splicing activities in vitro but also the trans-cleavage activities in cells of several ribozymes that targeted different sites. The ribozyme that could target the most accessible site identified by mapping studies was then the most active with high fidelity in cells as well as in vitro. These results demonstrate that the RNA mapping strategy represents an effective method to determine the accessible regions of target RNAs and have important implications for the development of various antiviral therapies which are based on RNA such as ribozyme, antisense, or siRNA.     

Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.

Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library. Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library. Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes. Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement. The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate. Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library.     

The structure and function of catalytic RNAs

Before the discovery of ribozymes, RNA had been proposed to function as a catalyst, based on the discovery that RNA folded into high-ordered structures as protein did. This hypothesis was confirmed in the 1980s, after the discovery of Tetrahymena group I intron and RNase P ribozyme. There have been about ten ribozymes identified during the past thirty years, as well as the fact that ribosomes function as ribozymes. Advances have been made in understanding the structures and functions of ribozymes, with numerous crystal structures resolved in the past years. Here we review the structure-function relationship of both small and large ribozymes, especially the structural basis of their catalysis. ribozyme, structure, catalysis Supported by National Natural Science Foundation of China (Grant No. 30330170) and National Key Basic Research and Development Program of China (Grant No. 2005CB724604)     

Functional identification of catalytic metal ion binding sites within RNA

The viability of living systems depends inextricably on enzymes that catalyze phosphoryl transfer reactions. For many enzymes in this class, including several ribozymes, divalent metal ions serve as obligate cofactors. Understanding how metal ions mediate catalysis requires elucidation of metal ion interactions with both the enzyme and the substrate(s). In the Tetrahymena group I intron, previous work using atomic mutagenesis and quantitative analysis of metal ion rescue behavior identified three metal ions (MA, MB, and MC) that make five interactions with the ribozyme substrates in the reaction's transition state. Here, we combine substrate atomic mutagenesis with site-specific phosphorothioate substitutions in the ribozyme backbone to develop a powerful, general strategy for defining the ligands of catalytic metal ions within RNA. In applying this strategy to the Tetrahymena group I intron, we have identified the pro-SP phosphoryl oxygen at nucleotide C262 as a ribozyme ligand for MC. Our findings establish a direct connection between the ribozyme core and the functionally defined model of the chemical transition state, thereby extending the known set of transition-state interactions and providing information critical for the application of the recent group I intron crystallographic structures to the understanding of catalysis.     

Ribozymes in the age of molecular therapeutics

Ribozymes are RNA molecules capable of sequence-specific cleavage of other RNA molecules. Since the discovery of the first group I intron ribozyme in 1982, new classes of ribozymes, each with their own unique reaction, target site specifications, and potential applications, have been identified. These include hammerhead, hairpin, hepatitis delta, varkud satellite, groups I and II intron, and RNase P ribozymes, as well as the ribosome and spliceosome. Meanwhile, ribozyme engineering has enabled the in vitro selection of synthetic ribozymes with unique properties. This, along with advances in ribozyme delivery methods and expression systems, has led to an explosion in the potential therapeutic applications of ribozymes, whether for anti-cancer or anti-viral therapy, or for gene repair.