Extended target-site specificity for a hammerhead ribozyme

Perriman, Rhonda; Delves, Angela; Gerlach, W. L.

doi:10.1016/0378-1119(92)90391-2

Cited by 114 publications

(82 citation statements)

References 20 publications

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“…However, it can be concluded that for each set of target RNAs, the naturally occurring motif GUC4 was always cleaved with a relative high efficiency. It is striking that AUCt was cleaved best in the current study, whereas it was not cleaved at all in the sequence context that Perriman et al (11) used. With regard to the sequence context, it may be worthwhile to note that the AUCt motif described here was part of the palindrome of the ClaI recognition sequence (AT/CGAT).…”

Section: Discussioncontrasting

confidence: 47%

“…Especially the relative cleavage rates within one set of target RNAs, should not be influenced by differences in helix H. Only the study of Koizumi et al (9) may be exceptional since they utilized hammerhead ribozymes based on the RNA transcript of newt satellite 2 DNA which later was found to cleave via alternative modes, including a double-hammerhead (44), so that comparison may be difficult. Similar to this study, Ruffner et al (10) and Perriman et al (11) had used pre-annealed ribozyme/substrate complexes, which were cleaved according to pseudo-first order kinetics, so that differences in hybridization or Km values are not relevant for the variation in detected cleavage. The relative cleavage rates of all four studies, standardized to GUC4 = 100%, are summarized in Table 1, which also indicates the neighboring nucleotides 16.3 and 1.1.…”

Section: Discussionmentioning

confidence: 89%

“…motifs, there are three previous studies in which NUC4. and GUXt motifs have been compared in the same sequence context (9)(10)(11). For each set of experiments another catalytic domain had been used.…”

Section: Discussionmentioning

confidence: 99%

“…This motif has been converted to AUC4, CUC4, UUC4, GUA4 or GUUt each by Koizumi et al and Ruffner et al (9,10), without loss of activity, even though the cleavage efficiencies varied among the motifs and were generally lower compared with the GUC4 motif. These studies allowed the conclusion that a consensus sequence 'UX4' or 'NUX ' can be described wherein N can be any nucleotide and X any but G. In a similar study, Perriman et al (11) …”

Section: Introductionmentioning

confidence: 92%

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Comparative analysis of cleavage rates after systematic permutation of the NUX↓ consensus target motif for hammerhead ribozymes

Zoumadakis

Tabler

1995

Nucl Acids Res

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Section: Discussioncontrasting

confidence: 47%

Section: Discussionmentioning

confidence: 89%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 92%

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Comparative analysis of cleavage rates after systematic permutation of the NUX↓ consensus target motif for hammerhead ribozymes

Zoumadakis

Tabler

1995

Nucl Acids Res

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“…This was surprising given the initial bias in the library (Fig 1a) and the fact that GUC is considered to be the most efficient and most common naturally occurring triplet. 25,26 Ribozyme cleavage activity against selected sites in vitro…”

Section: Rna Cleavage Detection By La-pcrmentioning

confidence: 99%

E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes

et al. 2003

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E6AP was originally identified as the ubiquitin-protein ligase involved in human papillomavirus (HPV) E6-mediated p53 degradation and has since been shown to act as an E3 ubiquitin-protein ligase in the ubiquitination of several other protein substrates. To further define E6AP function at the molecular and cellular levels, a ribozyme-based gene inactivation approach was adopted. A library of hammerhead ribozymes, with randomized arm sequences, was used to screen active molecules along the entire E6AP transcript for ribozyme-cleavable sites. Ligation-anchored PCR was adapted to detect cleavage products, and ribozymes designed to the selected sites were characterized both in vitro and in vivo. Ribozyme-mediated reduction in E6AP expression was found to enhance the apoptotic response of HeLa cells to mitomycin C-induced DNA damage. These findings suggest that E6AP has potential as a drug target, as its suppression can potentiate apoptosis in HPV-positive cells treated with a cytotoxic drug. Cancer Gene Therapy (2003) 10, 707-716. doi:10.1038/sj.cgt.7700623Keywords: E6AP; gene function; apoptosis; ribozyme; in vitro selection T he ubiquitin-dependent proteolytic pathway has emerged as a crucial mechanism in cellular regulation. 1 Protein ubiquitination involves a cascade of cellular enzymes called E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin-protein ligase). Ubiquitin is activated at its C-terminal glycine to form a thiolester with the active site cysteine on E1 in an ATP-dependent reaction. The activated ubiquitin is then transferred to one of a family of E2s, preserving the high-energy thiolester bond. E2 enzymes catalyze isopeptide bond formation between ubiquitin and substrate protein directly or in association with an E3. Multiubiquitinated proteins are recognized by a large multisubunit protease complex, the 26S proteosome and degraded.The 100 kDa cellular protein E6AP, in association with human papillomavirus (HPV) E6, was initially identified as a ubiquitin-protein ligase responsible for p53 degradation. 2 It has since been shown that E6AP acts as an E3 in the ubiquitin-dependent proteolysis of various other cellular proteins, both in the presence and absence of HPV E6. 3-7 E6AP was also the founding member of a family of structurally related E3s carrying a carboxyl terminus that is highly conserved among various organisms, termed the hect domain (homology to E6AP C terminus). 8 Besides its protein ligase function, E6APwas recently shown to act as a coactivator in nuclear hormone receptor-mediated transactivation, 9 and mutations in E6AP have been causally linked with a genetic neurological disorder known as Angelman Syndrome. 10,11 Despite the identification of several E6AP-interacting proteins, the role of E6AP in cellular physiology remains poorly understood. Recent works from several groups suggest that E6AP may be involved in the regulation of important cellular processes such as transcription, signal transduction, cell survival and/or apoptosis, cell-cycle contr...

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Retrovirus-mediated transfer of anti-MRD1 hammerhead ribozymes into multidrug-resistant human leukemia cells: Screening for effective target sites

et al. 1999

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Extended target-site specificity for a hammerhead ribozyme

Cited by 114 publications

References 20 publications

Comparative analysis of cleavage rates after systematic permutation of the NUX↓ consensus target motif for hammerhead ribozymes

Comparative analysis of cleavage rates after systematic permutation of the NUX↓ consensus target motif for hammerhead ribozymes

E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes

Retrovirus-mediated transfer of anti-MRD1 hammerhead ribozymes into multidrug-resistant human leukemia cells: Screening for effective target sites

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