Relationships between the activities in vitro and in vivo of various kinds of ribozyme and their intracellular localization in mammalian cells.
Nineteen different functional RNAs were synthesized for an investigation of the actions of ribozymes, in vitro and in vivo, under the control of two different promoters, tRNA or U6, which localize transcripts either in the cytoplasm or in the nucleus. No relationships were found between the activities of these RNAs in cultured cells and the kinetic parameters of their respective chemical cleavage reactions in vitro, indicating that in no case was chemical cleavage the rate-limiting step in vivo. For example, a hepatitis delta virus (HDV) ribozyme, whose activity in vitro was almost 3 orders of magnitude lower than that of a hammerhead ribozyme, still exhibited similar activity in cells when an appropriate expression system was used. As expected, external guide sequences, the actions of which depend on nuclear RNase P, were more active in the nucleus. Analysis of data obtained with cultured cells clearly demonstrated that the cytoplasmic ribozymes were significantly more active than the nuclear ribozymes, suggesting that mature mRNAs in the cytoplasm might be more accessible to antisense molecules than are pre-mRNAs in the nucleus. Our findings should be useful for the future design of intracellularly active functional molecules.
The Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology, 1-1-4 Higashi, Tsukuba Science City 305-8562, Japan.
Y
Y
Kato
T
T
Kuwabara
M
M
Warashina
H
H
Toda
K
K
Taira
D001483:Base Sequence; D003593:Cytoplasm / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D006868:Hydrolysis; D017403:In Situ Hybridization; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D016337:RNA, Catalytic; D012333:RNA, Messenger