The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization.
In order to better understand the influence of RNA transcript context on RNA localization and catalytic RNA efficacy in vivo, we have constructed and characterized several expression cassettes useful for transcribing short RNAs with well defined 5' and 3' appended flanking sequences. These cassettes contain promoter sequences from the human U1 snRNA, U6 snRNA, or tRNA Meti genes, fused to various processing/stabilizing sequences. The levels of expression and the sub-cellular localization of the resulting RNAs were determined and compared with those obtained from Pol II promoters normally linked to mRNA production, which include a cap and polyadenylation signal. The tRNA, Ul, and U6 transcripts were nuclear in localization and expressed at the highest levels, while the standard Pol II promoted transcripts were cytoplasmic and present at lower levels. The ability of these cassettes to confer ribozyme activity in vivo was tested with two assays. First, an SIV-growth hormone reporter gene was transiently transfected into human embryonic kidney cells expressing an anti-SIV ribozyme. Second, cultured T lymphocytes expressing an anti-HIV ribozyme were challenged with HIV. In both cases, we found that the ribozymes were effective only when expressed as capped, polyadenylated RNAs transcribed from Pol II cassettes that generate a cytoplasmically localized ribozyme that facilitates co-localization with its target. We also show that the inability of the other cassettes to support ribozyme-mediated inhibitory activity against their cytoplasmic target is very likely due to the resulting nuclear localization of these ribozymes. These studies demonstrate that the ribozyme expression cassette determines its intracellular localization and, hence, its corresponding functional activity.
Bertrand
E
E
Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Castanotto
D
D
Zhou
C
C
Carbonnelle
C
C
Lee
N S
NS
Good
P
P
Chatterjee
S
S
Grange
T
T
Pictet
R
R
Kohn
D
D
Engelke
D
D
Rossi
J J
JJ
D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D019556:COS Cells; D002478:Cells, Cultured; D003001:Cloning, Molecular; D000229:Dependovirus / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D015756:Genes, tat; D005822:Genetic Vectors / Q000502:physiology*; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D017403:In Situ Hybridization; D007668:Kidney / Q000166:cytology; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics / Q000378:metabolism; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000235:genetics; D012358:RNA, Transfer, Met / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D015302:Simian Immunodeficiency Virus / Q000235:genetics; D013347:Subcellular Fractions / Q000378:metabolism*; D013601:T-Lymphocytes
D016337:RNA, Catalytic; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D012358:RNA, Transfer, Met; C039919:RNA, recombinant; D012313:RNA