Résumé
Subcellular localization signals for several mRNAs
are positioned in their 3′ untranslated regions (UTR).
We have utilized the human α- and β-actin 3′
UTRs as signals for colocalizing hammerhead ribozymes with
a lacZ target mRNA. Ribozyme and target genes
containing matched or unmatched 3′ UTRs were cotransfected
into 12-day-old chicken embryonic myoblast and fibroblast
(CEMF) cultures and assayed by in situ hybridization
(ISH) using a dual label, antibody sandwich procedure,
and dual fluorescence microscopy to monitor intracellular
colocalization. β-galactosidase localization in transfectants
was visualized by incubation with X-gal and also quantitated
by an o-nitrophenyl β-D-galactopyranoside
(ONPG) assay. We found that the percentage of colocalization
using the matched α- or β-actin 3′ UTR (α–α
or β–β) was enhanced approximately threefold
relative to unmatched 3′ UTRs. The increase in ribozyme-mediated
inhibition of β-galactosidase activity observed when
matched 3′ UTRs were used was consistent with the
observed percentage of colocalization. These results represent
the first direct demonstration that mRNA localization signals
(zipcodes) can be utilized to enhance intracellular ribozyme
efficacy.