Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3' end can be exported efficiently and specifically to the cytoplasm in mammalian cells.
Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNA(Val)-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3' end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5' and 3' ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells.
Kuwabara
T
T
Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-4 Higashi, Tsukuba Science City 305-8562, Japan.
Warashina
M
M
Koseki
S
S
Sano
M
M
Ohkawa
J
J
Nakayama
K
K
Taira
K
K
D001483:Base Sequence; D001692:Biological Transport; D002467:Cell Nucleus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D003593:Cytoplasm / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012320:RNA Polymerase III / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000235:genetics; D012366:RNA, Transfer, Val / Q000235:genetics*
D016337:RNA, Catalytic; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D012358:RNA, Transfer, Met; D012366:RNA, Transfer, Val; C405486:U6 small nuclear RNA; D012320:RNA Polymerase III