Ribozymes: the characteristics and properties of catalytic RNAs

Abstract

Ribozymes, or catalytic RNAs, were discovered a little more than 15 years ago. They are found in the organelles of plants and lower eukaryotes, in amphibians, in prokaryotes, in bacteriophages, and in viroids and satellite viruses that infect plants. An example is also known of a ribozyme in hepatitis delta virus, a serious human pathogen. Additional ribozymes are bound to be found in the future, and it is tempting to regard the RNA component(s) of various ribonucleoprotein complexes as the catalytic engine, while the proteins serve as mere scaffolding^an unheard-of notion 15 years ago! In nature, ribozymes are involved in the processing of RNA precursors. However, all the characterized ribozymes have been converted, with some clever engineering, into RNA enzymes that can cleave or modify targeted RNAs (or even DNAs) without becoming altered themselves. While their success in vitro is unquestioned, ribozymes are increasingly used in vivo as valuable tools for studying and regulating gene expression. This review is intended as a brief introduction to the characteristics of the different identified ribozymes and their properties. ß

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

“…The presence of a poly(A) tail is generally thought to be a critical determinant of mRNA stability+ For some mRNAs, poly(A) shortening or removal is the ratedetermining event in their decay, whereas, for others, it may be an obligate event in their degradation, but it is not the rate-determining step (Chen et al+, 1995;Caponigro & Parker, 1996;+ The presence of a poly(A) tail provides an mRNA with a binding site for poly(A)-binding protein, which, in turn, serves to promote translational initiation and antagonize mRNA decapping in the cytoplasm (Gallie et al+, 1989;Caponigro & Parker, 1995;Tarun & Sachs, 1996)+ In this study, the TRP4-ribozyme allele produces a transcript lacking a poly(A) tail and, as a consequence, it would be predicted that this mRNA would be susceptible to nucleolytic attack and considerably less abundant than its wild-type counterpart+ In contrast, we found that the nonpolyadenylated ribozyme transcript accumulated to levels indicative of little, if any, change in stability+ Maintenance of the stability of the poly(A)-deficient TRP4-ribozyme mRNA is likely to be a result of the action of the ribozyme in generation of the mRNA 39 end, and not to some internal TRP4 sequence features, as deletion of the TRP4 39 UTR resulted in a significant decrease in the amount of TRP4 mRNA+ In support of this idea, a previous study determined that the presence of a ribozyme in an mRNA is not sufficient to render it more labile (Donahue & Fedor, 1997)+ Several plausible explanations could account for the unchanged stability of TRP4-ribozyme mRNA+ The secondary structure formed by the hammerhead ribozyme, or the terminal 29-39cyclic phosphate (Tanner, 1999), instead of a 39-OH, might impede the accessibility of 39 exonucleases to the mRNA+ Alternatively, the absence of a poly(A) tail might uncouple an otherwise linked mechanism in which decapping is triggered by deadenylation (Muhlrad et al+, 1994)+ A more likely explanation is that degradation of the TRP4 mRNA is normally associated with its translation (Jacobson & Peltz, 1996)+ In a manner previously exemplified by the MATa1 mRNA (Parker & Jacobson, 1990), impaired translation of the TRP4-ribozyme mRNA (see below) may reduce the extent to which ribosome-associated factors act to promote decay+…”

“…termini generated by hammerhead cleavage to serve as substrates for poly(A) polymerase (Tanner, 1999;Zhao et al+, 1999)+ In yeast, it has been possible to obtain polyadenylation of a ribozyme-containing transcript (Egli & Braus, 1994), an event that most likely depends on RNA sequence context, that is, triggering cryptic cleavage/polyadenylation signals+ Because we were unable to detect any polyadenylated transcripts in RACE experiments we conclude that the TRP4-ribozyme mRNA does not contain any cryptic signals+ Although mRNA 39-end processing and nuclear export have been shown to be linked (Eckner et al+, 1991;Hilleren et al+, 2001), the TRP4-ribozyme mRNA appears to be efficiently exported from the nucleus+ It is unclear whether this occurs by pathways previously shown to be effective for nonpolyadenylated RNAs like rRNA and tRNAs (Moy & Silver, 1999;Sarkar et al+, 1999;Simos & Hurt, 1999) or by some other mechanism+ Unlike metazoans and their unadenylated histone mRNAs, there are no known examples of yeast RNA polymerase II transcripts that lack poly(A) tails (Goffeau et al+, 1996)+ Future studies to assess the mechanism by which such nonpolyadenylated mRNAs are exported will be of interest+…”

“…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 ...…”

tRNA-dependent cleavage of the ColE1 plasmid-encoded RNA I

“…Examples are the activities of siRNA and microRNAs (Zhang et al, 2002;Doi et al, 2003;Zeng et Goodman, 2004; Yekta et al, 2004;Kim et al, 2005b;Behlke, 2006;Ronemus et al, 2006;Valencia-Sanchez et al, 2006). Nevertheless, other reports have described reactions of direct RNA-mediated cleavage of RNA (Tanner, 1999;Lilley, 1999;Doudna & Cech, 2002;Sago et al, 2004). These reactions include self-cleaving of RNA [for example:…”

2-Aminopurine as a real-time probe of enzymatic cleavage and inhibition of hammerhead ribozymes

“…1 Their functional diversity can be attributed to the complex three-dimensional folds that RNA can assume via a multitude of secondary structures and tertiary interactions. 2,3 Ribozymes, therefore, provide a unique opportunity to correlate RNA structure and function.…”