Ribozyme-mediated RNA degradation in nuclei suspension

Heidenreich, Olaf; Kang, Shin-Heh; Brown, David A.; Xu, Xiao; Swiderski, Piotr; Rossi, John J.; Eckstein, Fritz; Nerenberg, Michael

doi:10.1093/nar/23.12.2223

Cited by 46 publications

(22 citation statements)

References 37 publications

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“…Certain proteins have been reported to accelerate hammerhead ribozyme cleavage reactions [ 16 -181 and proteins present in nuclei suspension have also been shown to have the same effect [19]. These proteins, acting as chaperones, may facilitate the folding of a ribozyme into an active conformation intracellularly and thereby improve the cleavage activity.…”

Section: Discussionmentioning

confidence: 99%

Intermolecular Cleavage by the Newt Ribozyme

Marusic

Luzi

Barsacchi

et al. 1997

European Journal of Biochemistry

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To analyse the trans-cleavage activity of the hammerhead ribozyme occurring in the ovary of the newt (Notophthalmus, Triturus) in more detail, six synthetic ribozymes representing natural and modified hammerhead sequences were tested with both short oligoribonucleotides and long transcripts as substrates. The same analysis was also performed with the monomer (330 nucleotides) newt ribozyme and variants thereof. None of the ribozymes comprising the newt natural sequence showed activity under multiple turnover conditions, regardless of sequence changes in stem and loop 11. With excess of ribozyme, the same ribozymes cleaved only to a limited extent a short substrate and extremely poorly a target site embedded within a long transcript. The addition of whole ovary cell extract had little influence on cleavage activity of short substrates. However, sequence changes in stems I and 111 to target different sequences considerably improved cleavage ability of the ribozymes under all conditions used. An RNA secondarystructure folding program showed that ribozymes with the natural newt sequence did not fold in a hammerhead structure whereas those with the changes in stem I and 111 did. These results suggest that the sequence of the stems I and 111 impairs the assembly of the newt ribozyme into a bimolecular hammerhead complex in vitro and that proteins present in the ovaries do not facilitate activity.Keywords: catalytic RNA ; hammerhead ; trans-cleavage; RNA-binding protein.A family of highly repetitive DNA sequences, dispersed in small clusters in the newt (Notophthalmus, Triturus) genome, codes for stable, strand-specific transcripts found in both somatic and germinal tissues ([l] and Cremisi, F., personal communication). The transcripts correspond in size precisely to monomeric (330 bp) and multimeric DNA repeat units. Evidence that synthetic dimer-rize transcripts undergo site-specific, selfcatalyzed cleavage in vitro suggested that the same reaction might be involved in the processing of large multimeric transcripts in vivo 12, 31. The self-cleavage reaction requires Mg" and occurs within a conserved structure named the hammerhead, similar to that of infectious plant RNAs [4].The ovarian ribozyme transcripts differ from the analogous transcripts in other newt tissues in having an intact self-cleavage site, located about SO nucleotides from the 5' end [2]. Therefore, it seems that monomer length transcripts of the ovarian newt ribozyme ,are more likely to arise rather from trancription than from self-processing by cleavage. It has indeed been shown that the same promoter elements that regulate polymerase-11-dependent transcription of small nuclear RNAs are involved in transcription of the newt ribozyme [5, 61. The presence of an apparently functional gene that codes for the small RNA with a hammerhead domain suggests that a biological function of this particular ribozyme may be trans-cleavage of a specific, but still unknown RNA target in vivo [5, 61. Here we report an in vitro study of the trans-cleaving properties o...

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Section: Discussionmentioning

confidence: 99%

Intermolecular Cleavage by the Newt Ribozyme

Marusic

Luzi

Barsacchi

et al. 1997

European Journal of Biochemistry

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“…RNAs used for in vitro selection were produced by in vitro transcription with T7 RNA polymerase in presence of 2Ј-fluoromodified pyrimidine nucleotide triphosphates (TriLink BioTechnologies, Inc., San Diego, CA), together with unmodified purine ribonucleotides in an optimized transcription buffer (30). The 2Ј-F-RNA transcripts were purified by electrophoresis on a 10% (w/v) denaturing polyacrylamide gel in TBE buffer.…”

Section: Methodsmentioning

confidence: 99%

Characterization of 2′-Fluoro-RNA Aptamers That Bind Preferentially to Disease-associated Conformations of Prion Protein and Inhibit Conversion

Rhie

Kirby

Sayer

et al. 2003

Journal of Biological Chemistry

173

148

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We have isolated artificial ligands or aptamers for infectious prions in order to investigate conformational aspects of prion pathogenesis. The aptamers are 2 -fluoro-modified RNA produced by in vitro selection from a large, randomized library. One of these ligands (aptamer SAF-93) had more than 10-fold higher affinity for PrP Sc than for recombinant PrP C and inhibited the accumulation of PrP res in near physiological cell-free conversion assay. To understand the molecular basis of these properties and to distinguish specific from nonspecific aptamer-PrP interactions, we studied deletion mutants of bovine PrP in denatured, ␣-helix-rich and ␤-sheet-rich forms. We provide evidence that, like scrapie-associated fibrils (SAF), the ␤-oligomer of PrP bound to SAF-93 with at least 10-fold higher affinity than did the ␣-form. This differential affinity could be explained by the existence of two binding sites within the PrP molecule. Site 1 lies within residues 23-110 in the unstructured N terminus and is a nonspecific RNA binding site found in all forms of PrP. The region between residue 90 and 110 forms a hinge region that is occluded in the ␣-rich form of PrP but becomes exposed in the denatured form of PrP. Site 2 lies in the region C-terminal of residue 110. This site is ␤-sheet conformation-specific and is not recognized by control RNAs. Taken together, these data provide for the first time a specific ligand for a disease conformation-associated site in a region of PrP critical for conformational conversion. This aptamer could provide tools for the further analysis of the processes of PrP misfolding during prion disease and leads for the development of diagnostic and therapeutic approaches to TSEs.

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“…25. Briefly, transcription by T7 RNA polymerase (New England Biolabs) was incubated at 37°C for at least 6 h in 40 mM Tris, pH 8.1, 6 mM magnesium chloride, 1 mM spermidine, 5 mM dithiothreitol, and 1 mM NTPs.…”

Section: Methodsmentioning

confidence: 99%

Structural Determinants of Conformationally Selective, Prion-binding Aptamers

Sayer¹,

Cubin

Rhie

et al. 2004

Journal of Biological Chemistry

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We have recently described the isolation of 2-fluoropyrimidine-substituted RNA aptamers that bind selectively to disease-associated ␤-sheet-rich forms of the prion protein, PrP, from a number of mammalian species. These aptamers inhibit the accumulation of protease-resistant forms of PrP in a prion-seeded, in vitro conversion assay. Here we identify the minimal portions of two of these aptamers that retain binding specificity. We determine their secondary structures by a combination of modeling and solution probing. Finally, we identify an internal site for biotinylation of a minimized, synthetic aptamer and use the resultant reagent in the detection of abnormal forms of PrP in vitro.

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Ribozyme-mediated RNA degradation in nuclei suspension

Cited by 46 publications

References 37 publications

Intermolecular Cleavage by the Newt Ribozyme

Intermolecular Cleavage by the Newt Ribozyme

Characterization of 2′-Fluoro-RNA Aptamers That Bind Preferentially to Disease-associated Conformations of Prion Protein and Inhibit Conversion

Structural Determinants of Conformationally Selective, Prion-binding Aptamers

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