mRNA localization signals can enhance the intracellular effectiveness sf hammerhead ribozymes

Subcellular localization signals for several mRNAs are positioned in their 3' untranslated regions (UTR), We have utilized the human alpha- and beta-a ctin 3' UTRs as signals for colocalizing hammerhead ribozymes with a lacZ t arget mRNA, Ribozyme and target genes containing matched or unmatched 3' UT Rs were cotransfected into 12-day-old chicken embryonic myoblast and fibrob last (CEMF) cultures and assayed by in situ hybridization (ISH) using a dua l label, antibody sandwich procedure, and dual fluorescence microscopy to m onitor intracellular colocalization. beta-galactosidase localization in tra nsfectants was visualized by incubation with X-gal and also quantitated by an o-nitrophenyl beta-D-galactopyranoside (ONPG) assay, We found that the p ercentage of colocalization using the matched alpha- or beta-actin 3' UTR ( alpha-alpha or beta-beta) was enhanced approximately threefold relative to unmatched 3' UTRs, The increase in ribozyme-mediated inhibition of beta-gal actosidase 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 b e utilized to enhance intracellular ribozyme efficacy.