Identification of genes by hybrid ribozymes that couple cleavage activity with the unwinding activity of an endogenous RNA helicase.
Novel ribozymes that couple the cleavage activity of hammerhead ribozymes with the unwinding activity of RNA helicase eIF4AI were constructed. This leads to extremely efficient cleavage of the target mRNA, regardless of the secondary structure of the RNA, and eliminates one of the major problems: many target sites on the RNA were previously inaccessible to cleavage due to secondary and/or tertiary structure formation. Moreover, libraries of hybrid ribozymes with randomized binding arms were introduced into cells. This procedure made it possible to readily identify the relevant genes associated with phenotype. Specifically, four genes known to be in the Fas-mediated apoptosis pathway were identified along with additional genes. This application of a randomized library of hybrid ribozymes represents a simple, powerful method for the identification of genes associated with specific phenotypes in the post-genome era.
Kawasaki
Hiroaki
H
Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan.
Taira
Kazunari
K
D048868:Adaptor Proteins, Signal Transducing; D000241:Adenosine / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D039601:Eukaryotic Initiation Factor-4A / Q000378:metabolism; D053200:Fas-Associated Death Domain Protein; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011108:Polymers / Q000378:metabolism; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism*; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012366:RNA, Transfer, Val / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D019014:fas Receptor / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D011108:Polymers; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012366:RNA, Transfer, Val; D011994:Recombinant Proteins; D019014:fas Receptor; C069424:polyadenosine; D039601:Eukaryotic Initiation Factor-4A; D020365:RNA Helicases; D000241:Adenosine