Functional colocalization of ribozymes and target mRNAs in Drosophila oocytes.
The effectiveness of catalytic RNAs (ribozymes) should be increased when they are colocalized to the same intracellular compartment as their RNA targets. We colocalized ribozymes with their mRNA targets in an animal model by using the discrete RNA localization signals present in the 3' untranslated regions (UTRs) of Drosophila bicoid and oskar mRNAs. These signals have been fused to a lacZ mRNA target and hammerhead ribozymes targeted against lacZ. Ribozyme efficacy was first assessed by an oligodeoxyribonucleotide-based assay to identify the most accessible sites for ribozyme interaction on native lacZ transcripts in ovary extracts. The most accessible sequence was used for the design and in vivo testing of a hammerhead ribozyme. When the ribozyme and target with synonymous 3' UTRs were expressed in the same ovaries, colocalization could be indirectly demonstrated by in situ hybridization. Colocalized ribozyme and target mRNAs resulted in a two- to threefold enhancement of ribozyme function compared with noncolocalized transcripts. This study provides the first demonstration of functional ribozyme target colocalization in an animal model.
Lee
N S
NS
Department of Molecular Biology, Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Sun
B
B
Williamson
R
R
Gunkel
N
N
Salvaterra
P M
PM
Rossi
J J
JJ
D020413:3' Untranslated Regions / Q000235:genetics / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D004330:Drosophila / Q000235:genetics*; D029721:Drosophila Proteins; D005260:Female; D018398:Homeodomain Proteins / Q000235:genetics; D017403:In Situ Hybridization; D019476:Insect Proteins / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D010053:Ovary / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D020413:3' Untranslated Regions; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D019476:Insect Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D015534:Trans-Activators; C056478:bcd protein, Drosophila; C091675:osk protein, Drosophila; D001616:beta-Galactosidase