C-SPACE (cleavage-specific amplification of cDNA ends): a novel method of ribozyme-mediated gene identification

• Published in: Nucleic acids research Vol. 29; no. 19; pp. E94 - e94
• Main Authors: Krüger, M, Beger, C, Welch, P J, Barber, J R, Wong-Staal, F
• Format: Journal Article
• Language: English
• Published: England Oxford Publishing Limited (England) 01.10.2001; Oxford University Press
• Subjects: 5' Untranslated Regions; Base Sequence; Binding Sites; Cysteine Endopeptidases - genetics; DNA, Complementary; Genes; HeLa Cells; Hepacivirus - genetics; Hepatitis C virus; Humans; Multienzyme Complexes - genetics; NAR Methods Online; Polymerase Chain Reaction - methods; proteasome 20S; Proteasome Endopeptidase Complex; PSMA7 gene; RNA, Catalytic - metabolism; RNA, Messenger - metabolism; RNA, Viral - metabolism; Tumor Cells, Cultured
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/29.19.e94

A hairpin ribozyme, RzCR2A, directed against position 323 of the hepatitis C virus 5'-untranslated region (HCV 5'-UTR) was used to establish and validate a novel method for the detection of cellular target molecules for hairpin ribozymes, termed C-SPACE (cleavage-specific amplification of cDNA ends). For C-SPACE, HeLa mRNA containing the transcript of interest was subjected to in vitro cleavage by RzCR2A in parallel with a control ribozyme, followed by reverse transcription using a modified SMART cDNA amplification method and cleavage-specific PCR analysis. C-SPACE allowed identification of the RzCR2A target transcript from a mixture containing the entire cellular mRNA while only requiring knowledge of the ribozyme binding sequence for amplification. In a similar approach, C-SPACE was used successfully to identify human 20S proteasome alpha-subunit PSMA7 mRNA as the cellular target RNA of Rz3'X, a ribozyme originally designed to cleave the negative strand HCV 3'-UTR. Rz3'X was found to substantially inhibit HCV internal ribosome entry site (IRES) activity and PSMA7 was subsequently confirmed to be involved in HCV IRES-mediated translation. Thereby, C-SPACE was validated as a powerful tool to rapidly identify unknown target RNAs recognized and cleaved by hairpin ribozymes.