mRNA localization signals can enhance the intracellular effectiveness of hammerhead ribozymes.
Subcellular localization signals for several mRNAs are positioned in their 3' untranslated regions (UTR). We have utilized the human alpha- and beta-actin 3' UTRs as signals for colocalizing hammerhead ribozymes with a lacZtarget 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. Beta-galactosidase localization in transfectants was visualized by incubation with X-gal and also quantitated by an o-nitrophenyl beta-D-galactopyranoside (ONPG) assay. We found that the percentage 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-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.
Lee
N S
NS
Department of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010-3011, USA.
Bertrand
E
E
Rossi
J
J
D020413:3' Untranslated Regions / Q000235:genetics; D000199:Actins / Q000235:genetics / Q000648:ultrastructure; D000818:Animals; D002642:Chick Embryo; D004305:Dose-Response Relationship, Drug; D005347:Fibroblasts; D049452:Green Fluorescent Proteins; D006321:Heart / Q000196:embryology; D006801:Humans; D017403:In Situ Hybridization; D007763:Lac Operon / Q000235:genetics; D008164:Luminescent Proteins / Q000378:metabolism; D008957:Models, Genetic; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000235:genetics* / Q000494:pharmacology / Q000502:physiology / Q000648:ultrastructure; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D020413:3' Untranslated Regions; D000199:Actins; D008164:Luminescent Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase