Résumé
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 tRNAMeti 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, U1, 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.