Samarsky, D.A. et al. A small nucleolar RNA:ribozyme hybrid cleaves a nucleolar RNA target in vivo with near-perfect efficiency. Proc. Natl. Acad. Sci. USA 96, 6609-6614

Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/1999; 96(12):6609-14. DOI: 10.1073/pnas.96.12.6609
Source: PubMed


A hammerhead ribozyme has been localized to the yeast nucleolus by using the U3 small nucleolar RNA as a carrier. The hybrid small nucleolar RNA:ribozyme, designated a "snorbozyme," is metabolically stable and cleaves a target U3 RNA with nearly 100% efficiency in vivo. This is the most efficient in vivo cleavage reported for a trans-acting ribozyme. A key advantage of the model substrate featured is that a stable, trimmed cleavage product accumulates. This property allows accurate kinetic measurements of authentic cleavage in vivo. The system offers new avenues for developing effective ribozymes for research and therapeutic applications.

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Available from: Dmitry Samarsky, May 16, 2014
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    • "We therefore employed our Pol I minigene system to define the sequence requirements for T1 cleavage. We prepared constructs in which the Rnt1 cleavage site is substituted with a hammerhead ribozyme (20) (ΔRnt1 + RZ) or its mutant inactive form (ΔRnt1 + RZmut). Similarly, to study the co-transcriptional cleavage at T1, we either deleted the T-rich tract (ΔT1) or substituted it with the WT or mutant ribozyme (ΔT1 + RZ, ΔT1 + RZmut). "
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    ABSTRACT: Ribosomal RNA, transcribed by RNA polymerase (Pol) I, accounts for most cellular RNA. Since Pol I transcribes rDNA repeats with high processivity and polymerase density, transcription termination is a critical process. Early in vitro studies proposed polymerase pausing by Reb1 and transcript release at the T-rich element T1 determined transcription termination. However recent in vivo studies revealed a 'torpedo' mechanism for Pol I termination: co-transcriptional RNA cleavage by Rnt1 provides an entry site for the 5'-3' exonuclease Rat1 that degrades Pol I-associated transcripts destabilizing the transcription complex. Significantly Rnt1 inactivation in vivo reveals a second co-transcriptional RNA cleavage event at T1 which provides Pol I with an alternative termination pathway. An intact Reb1-binding site is also required for Rnt1-independent termination. Consequently our results reconcile the original Reb1-mediated termination pathway as part of a failsafe mechanism for this essential transcription process.
    Nucleic Acids Research 10/2010; 39(4):1439-48. DOI:10.1093/nar/gkq894 · 9.11 Impact Factor
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    • "Since the degradation rate constant of the HHRz transcripts by the cellular RNA-degrading machinery (k DegPre ) is positively correlated with the stringency of the selection for faster-cleaving HHRzs, it may be important to embed pools in short-lived transcripts (which could in turn be engineered by the introduction of degradation signals) (Noonberg et al. 1996). Moreover, negative selections for allosteric ribozymes that do not cleave in the absence of their effectors should be carried out by transferring a pool to a more stable transcript (Samarsky et al. 1999). The model we have developed can now be used during directed evolution experiments to determine whether and how kinetic parameters of ribozymes in vivo can be selected. "
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    ABSTRACT: Selection may prove to be a powerful tool for the generation of functional RNAs for in vivo genetic regulation. However, traditional in vitro selection schemes do not mimic physiological conditions, and in vivo selection schemes frequently use small pool sizes. Here we describe a hybrid in vitro/in vivo selection scheme that overcomes both of these disadvantages. In this new method, PCR-amplified expression templates are transfected into mammalian cells, transcribed hammerhead RNAs self-cleave, and the extracted, functional hammerhead ribozyme species are specifically amplified for the next round of selection. Using this method we have selected a number of cis-cleaving hammerhead ribozyme variants that are functional in vivo and lead to the inhibition of gene expression. More importantly, these results have led us to develop a quantitative, kinetic model that can be used to assess the stringency of the hybrid selection scheme and to direct future experiments.
    RNA 09/2009; 15(11):2035-45. DOI:10.1261/rna.1635209 · 4.94 Impact Factor
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    • "The Rz sequence was inserted in the reverse orientation to specifically induce self-cleavage of antisense RNA. To test the efficiency of the ribozyme, we performed an aging experiment with strains in which the endogenous PHO84 gene had been replaced by a PHO84 gene containing either the wild-type (PHO84Rz) or a mutant ribozyme (PHO84Rzm) harboring a single base change in its catalytic site that abolishes self-cleavage (Fig. 5B; Samarsky et al. 1999; Lacadie et al. 2006). As shown earlier, PHO84 expression was silenced in the progeny of 25-d-old wild-type cells (Fig. 5B, lanes 1,2). "
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    ABSTRACT: Homology-dependent gene silencing, a phenomenon described as cosuppression in plants, depends on siRNAs. We provide evidence that in Saccharomyces cerevisiae, which is missing the RNAi machinery, protein coding gene cosuppression exists. Indeed, introduction of an additional copy of PHO84 on a plasmid or within the genome results in the cosilencing of both the transgene and the endogenous gene. This repression is transcriptional and position-independent and requires trans-acting antisense RNAs. Antisense RNAs induce transcriptional gene silencing both in cis and in trans, and the two pathways differ by the implication of the Hda1/2/3 complex. We also show that trans-silencing is influenced by the Set1 histone methyltransferase, which promotes antisense RNA production. Finally we show that although antisense-mediated cis-silencing occurs in other genes, trans-silencing so far depends on features specific to PHO84. All together our data highlight the importance of noncoding RNAs in mediating RNAi-independent transcriptional gene silencing.
    Genes & development 08/2009; 23(13):1534-45. DOI:10.1101/gad.522509 · 10.80 Impact Factor
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