Cis-cleavage affects hammerhead and hairpin ribozyme steady-state levels differently and has strong impact on trans-targeting efficiency.
Trans-cleaving hammerhead or hairpin ribozymes were expressed in transgenic mice and in cell lines, using a cassette containing a second cis-cleaving hammerhead ribozyme positioned 3' of the trans-cleaving hammerhead or hairpin ribozyme. Cis-cleavage could be detected readily in transgenic mice, demonstrating in vivo release of the desired short trans-cleaving ribozyme transcript with a defined 3'-end. In transgenic organs, all cis-cleavage products containing a hairpin ribozyme were found at significantly higher steady-state levels than products containing a hammerhead ribozyme. Furthermore, an organ difference - kidney > liver > lung > spleen - regarding steady-state levels of both 5' and 3' cleavage products was found. In pools of stably transfected human T cells (HUT78), the efficacy of the 3' cis-cleavage was found to affect both the steady-state level and the antiviral efficiency of a trans-cleaving hairpin ribozyme targeting HIV-1. Insertion of a point mutation, efficiently inhibiting the cis-cleavage mechanism, led to higher overall steady-state levels of the noncleaved full-length transcript but, at the same time, also abolished the hairpin ribozyme protection against HIV-1 infection. We conclude that the cis-cleavage affects hammerhead and hairpin ribozyme steady-state levels differently and that it has a strong impact on trans-targeting efficiency.
Andäng
Michael
M
Division of Molecular Neurobiology, Karolinska Institutet, MBB, 171 77 Stockholm, Sweden. michael.andang@mbb.ki.se
Maijgren-Steffensson
Catharina
C
Hinkula
Jorma
J
Ahrlund-Richter
Lars
L
D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D006801:Humans; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D008099:Liver / Q000201:enzymology; D008168:Lung / Q000201:enzymology; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology
D017931:DNA Primers; D016337:RNA, Catalytic