C-SPACE (cleavage-specific amplification of cDNA ends): a novel method of ribozyme-mediated gene identification.
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.
Krüger
M
M
University of California San Diego School of Medicine, 0665, 9500 Gilman Drive, La Jolla, CA 92093-0665, USA.
Beger
C
C
Welch
P J
PJ
Barber
J R
JR
Wong-Staal
F
F
D020121:5' Untranslated Regions; D001483:Base Sequence; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000235:genetics; D018076:DNA, Complementary; D005796:Genes; D006367:HeLa Cells; D016174:Hepacivirus / Q000235:genetics; D006801:Humans; D009097:Multienzyme Complexes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D046988:Proteasome Endopeptidase Complex; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020121:5' Untranslated Regions; D018076:DNA, Complementary; D009097:Multienzyme Complexes; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral; C418481:hairpin ribozyme; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex