SMALL CATALYTIC RNAs

Symons, Robert H.

doi:10.1146/annurev.bi.61.070192.003233

Cited by 429 publications

(168 citation statements)

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“…) than most natural self-cleaving ribozymes (17). Therefore, we subjected a mutagenized pool based on the original class II deoxyribozyme to additional rounds of in vitro selection to improve catalytic activity and to provide an artificial phylogeny of variant DNAs for comparative sequence analysis.…”

Section: Resultsmentioning

confidence: 99%

An amino acid as a cofactor for a catalytic polynucleotide

Roth

Breaker

1998

Proc. Natl. Acad. Sci. U.S.A.

221

168

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Natural ribozymes require metal ion cofactors that aid both in structural folding and in chemical catalysis. In contrast, many protein enzymes produce dramatic rate enhancements using only the chemical groups that are supplied by their constituent amino acids. This fact is widely viewed as the most important feature that makes protein a superior polymer for the construction of biological catalysts. Herein we report the in vitro selection of a catalytic DNA that uses histidine as an active component for an RNA cleavage reaction. An optimized deoxyribozyme from this selection requires L-histidine or a closely related analog to catalyze RNA phosphoester cleavage, producing a rate enhancement of Ϸ1-million-fold over the rate of substrate cleavage in the absence of enzyme. Kinetic analysis indicates that a DNA-histidine complex may perform a reaction that is analogous to the first step of the proposed catalytic mechanism of RNase A, in which the imidazole group of histidine serves as a general base catalyst. Similarly, ribozymes of the ''RNA world'' may have used amino acids and other small organic cofactors to expand their otherwise limited catalytic potential.The ''RNA world'' theory for the origins of life holds that RNA was able to replicate in the absence of DNA and proteins (1). Some manifestations of this theory (1-3) include the view that, during the latter stages of the RNA world, ribozymes were not primitive and ineffectual but were directing a complex metabolic state replete with RNA and DNA synthesis machinery and a host of RNA enzymes necessary to catalyze supportive chemical reactions. Proponents of this view argue that a sophisticated metabolism mediated entirely by RNA would have been necessary for ribozymes to give rise to encoded protein synthesis-a precursor to the complex system of translation that is present in all extant organisms. To achieve maximum catalytic rate enhancements, natural ribozymes require Mg 2ϩ or other metal ion cofactors to induce structure formation or to participate directly in the catalytic process (4), perhaps defining a limitation to the catalytic potential for polynucleotide-based enzymes. When the limited catalytic repertoire of natural ribozymes is considered, the notion that complex networks of metabolic reactions could be guided entirely by RNA appears tenuous. Ancient ribozymes, however, may have created a larger catalytic repertoire by using various divalent metals (5) and by using nucleotide-like cofactors (6-8) to supplement the limited set of chemical groups that make up unmodified RNA. In recent years, a surprising number of artificial ribozymes have been created by using in vitro selection (9) that catalyze various chemical reactions including alkylation, acylation, phosphorylation, and ester and amide bond formation. In addition, the catalytic rates of ribozymes can be tightly regulated by using allosteric mechanisms (10), indicating that the potential for extensive and sophisticated catalysis by nucleic acids is large. Moreover, the full range of reac...

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Section: Resultsmentioning

confidence: 99%

An amino acid as a cofactor for a catalytic polynucleotide

Roth

Breaker

1998

Proc. Natl. Acad. Sci. U.S.A.

221

168

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“…The 39 untranslated region (39 UTR) of an mRNA plays an important role in 39 end processing of eukaryotic transcripts+ Sequence motifs, located in the 39 UTR, are necessary for cleavage at a specific site and the addition of a poly(A) tail (for reviews, see Wahle & Rüegsegger, 1999;Zhao et al+, 1999)+ The sequence motifs of yeast transcripts that direct cleavage and polyadenylation are not as conserved as in higher eukaryotes+ Several yeast 39 untranslated regions were shown to carry degenerate motifs that can at least partially replace one another (Egli et al+, 1995;Guo et al+, 1995)+ Deletion of the 39 UTR usually results in a significant reduction of the amount of gene product+ A deletion of the 39 UTR of the TRP4 gene led to a reduced amount of gene product without affecting cellular growth rates (Düvel et al+, 1999)+ A large multiprotein complex performs cleavage and polyadenylation of pre-mRNAs in eukaryotes (Keller & Minvielle-Sebastia, 1997;Zhao et al+, 1999)+ A subset of these factors affects only the polyadenylation efficiency whereas others are necessary for both cleavage and polyadenylation+ A complex pattern of interactions connects the different components with each other and cooperative interactions between the processing factors have been observed (Kessler et al+, 1997)+ As a consequence, cleavage and polyadenylation are coupled processes in vivo+ However, an uncoupling of cleavage and polyadenylation was achieved in vivo in yeast by using an artificial hammerhead ribozyme as a mRNA 39 end processing signal (Egli & Braus, 1994)+ Ribozymes are RNAs with an enzymatic activity for RNA cleavage (Altman, 1990;Cech, 1990)+ Hammerhead ribozymes are a subset of self-cleaving ribozymes that were originally isolated from plant viroids (Symons, 1992)+ Hammerhead ribozymes consist of three stem-loop-forming regions and two highly conserved single-stranded sequences+ Cleavage occurs at a triplet sequence, usually GUC+ Hydrolysis of the phosphodiester bond results in a 29-39-cyclic phosphate and a 59-hydroxyl product (Tanner, 1999)+ This is in contrast to enzymatic cleavage by processing factors that generally result in a 39-hydroxyl and a 59-phosphate+ Because the catalytic action of ribozymes has been studied thoroughly, new ribozymes were constructed providing applications in biotechnology and medicine+ Trans-acting ribozymes, for example, cleave target mRNAs at specific cleavage motifs, and are widely used to decrease the amount of the mRNA of genes of interest (Birikh et al+, 1997;Tanner, 1999)+ The hammerhead ribozyme used in this study cleaves itself efficiently in yeast (Egli & Braus, 1994)+ This prompted us to determine whether a hammerhead ribozyme could replace the normal mechanism of 39 end formation in an authentic yeast transcript+ We have now found that replacing the 39 UTR of the TRP4 gene with a hammerhead ribozyme results in a ...…”

Section: Introductionmentioning

confidence: 99%

Replacement of the yeast TRP4 3??? untranslated region by a hammerhead ribozyme results in a stable and efficiently exported mRNA that lacks a poly(A) tail

Düvel

Valerius

Mangus

et al. 2002

RNA

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The mRNA poly(A) tail serves different purposes, including the facilitation of nuclear export, mRNA stabilization, efficient translation, and, finally, specific degradation. The posttranscriptional addition of a poly(A) tail depends on sequence motifs in the 39 untranslated region (39 UTR) of the mRNA and a complex trans-acting protein machinery. In this study, we have replaced the 39 UTR of the yeast TRP4 gene with sequences encoding a hammerhead ribozyme that efficiently cleaves itself in vivo. Expression of the TRP4-ribozyme allele resulted in the accumulation of a nonpolyadenylated mRNA. Cells expressing the TRP4-ribozyme mRNA showed a reduced growth rate due to a reduction in Trp4p enzyme activity. The reduction in enzyme activity was not caused by inefficient mRNA export from the nucleus or mRNA destabilization. Rather, analyses of mRNA association with polyribosomes indicate that translation of the ribozyme-containing mRNA is impaired. This translational defect allows sufficient synthesis of Trp4p to support growth of trp4 cells, but is, nevertheless, of such magnitude as to activate the general control network of amino acid biosynthesis.

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“…The discovery that certain RNA species possess catalytic activity has generated significant interest in the potential therapeutic use of catalytic RNA molecules (ribozymes) in controlling gene expression (for a review, see Christoffersen & Marr, 1995)+ Ribozymes have been shown to function in trans and can be directed against foreign target sequences by flanking the catalytic core with sequences complementary to the target (Uhlenbeck, 1987;Haseloff & Gerlach, 1988)+ The hammerhead is the smallest of the known ribozyme motifs and therefore amenable to experimental manipulation (for a review, see Symons, 1992)+ Hammerhead ribozymes have broad potential as therapeutic agents for the selective control of gene expression (for a review, see Haseloff & Gerlach, 1988;Sarver et al+, 1990;Christoffersen & Marr, 1995)+ An important problem confronting the use of hammerhead ribozymes as therapeutic agents is that of maximizing the interaction of ribozymes to their target RNAs in vivo+ Experiments employing the unique property of retroviruses to dimerize prior to and during packaging have provided a paradigm for ribozyme-target colocalization (Sullenger & Cech, 1993;Pal et al+, 1998)+ The dimerization and packaging of retroviral RNAs creates a unique physical association of two genomic RNAs+ When a ribozyme is tethered to the dimerization domain, the physical interaction of two dimerization sequences facilitates the base pairing of ribozyme to target+ Physical associations of nonviral RNAs occur within cells, but these usually involve specific base pairing interactions such as snRNAs with splicing signals (Wu & Manley, 1991;Sun & Manley, 1995;Incorvaia & Padgett, 1998)+ The interaction of U1 snRNA with the 59 splice signal has been used as an approach for colocalization of a ribozyme with an HIV target (Michienzi et al+, 1998)+ More subtle methods for ribozyme-target colocalization can take advantage of the properties of some messenger RNAs to be localized within specific subcellular compartments+ The first evidence for cytoplasmic mRNA localization came from the observation that actin tran-scripts are unevenly distributed in ascidian embryos (Jeffery et al+, 1983)+ Subsequently, several maternal mRNAs were identified in Xenopus (Melton, 1987) and Drosophila (Frigerio et al+, 1986) that are localized during oogenesis, and many mRNAs are localized in neurons (Garner et al+, 1988;Burgin et al+, 1990;Tiedge et al+, 1991) and oligodendrocytes (Ainger et al+, 1993)+ Localized mRNAs have also been discovered in somatic cells …”

Section: Introductionmentioning

confidence: 99%