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 constr ucted and characterized several expression cassettes useful for transc ribing short RNAs with well defined 5' and 3' appended flanking sequen ces. These cassettes contain promoter sequences from the human U1 snRN A, U6 snRNA, or tRNA(Meti) genes, fused to various processing/stabiliz ing sequences. The levels of expression and the sub-cellular localizat ion of the resulting RNAs were determined and compared with those obta ined from Pol II promoters normally linked to mRNA production, which i nclude a cap and polyadenylation signal. The tRNA, U1, and U6 transcri pts were nuclear in localization and expressed at the highest levels, while the standard Pol II promoted transcripts were cytoplasmic and pr esent at lower levels. The ability of these cassettes to confer ribozy me activity in vivo was tested with two assays. First, an SIV-growth h ormone reporter gene was transiently transfected into human embryonic kidney cells expressing an anti-SIV ribozyme. Second, cultured T lymph ocytes expressing an anti-HIV ribozyme were challenged with HIV. In bo th cases, we found that the ribozymes were effective only when express ed as capped, polyadenylated RNAs transcribed from Pol II cassettes th at generate a cytoplasmically localized ribozyme that facilitates co-l ocalization with its target. We also show that the inability of the ot her 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 ri bozyme expression cassette determines its intracellular localization a nd, hence, its corresponding functional activity.