Hammerhead ribozyme cleavage of apolipoprotein B mRNA generates a truncated protein.
Target substrate-specific hammerhead ribozyme cleaves the specific mRNA and results in the inhibition of gene expression. In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To modulate apoB gene expression, we designed hammerhead ribozymes targeted at AUA(6665) and GUA(6679) of apoB mRNA, designated RB16 and RB15, respectively, and investigated their effects on apoB mRNA in HepG2 cells. The results demonstrated that RB15 and RB16 ribozyme RNAs cleaved apoB RNA efficiently in vitro. Both ribozymes, RB15 and RB16, were used to construct recombinant adenoviral vectors, designated AvRB15 and AvRB16, respectively, for in vivo gene transfer. HepG2 cells were infected with 2 x 10(5) plaque-forming units of AvRB15 for 5, 10, 15, and 24 h. An RNase protection assay showed that the expression of the RB15 transcript was time-dependent; it increased approximately 300-fold from 5 to 24 h. Using reverse ligation-mediated polymerase chain reaction, the 3' cleavage product of apoB mRNA was detected, and the exact cleavage site of apoB mRNA was confirmed by sequencing. Importantly, the levels of apoB mRNA in HepG2 cells decreased approximately 80% after AvRB15 infection. Pulse/chase experiments on HepG2 cells treated with AvRB15 and AvRB16 demonstrated that ribozyme cleavage produced a truncated protein that was secreted at a density of 1. 063-1.210 g/ml. The cleavage activity of RB15 on apoB mRNA was more efficient than that of RB16. Moreover, pulse/chase experiments in HepG2 cells treated with AvRB15 revealed that most of the truncated apoB protein was degraded intracellularly. We conclude that hammerhead ribozyme targeted at GUA(6679) of apoB mRNA cleaves apoB mRNA, results in decreased apoB mRNA levels, and generates a truncated apoB of the expected size in vivo. Thus, the therapeutic application of ribozyme in regulating apoB production holds promise.
Wang
J P
JP
Departments of Medicine and Cell Biology, Baylor College of Medicine, Houston, Texas 77030, USA.
Enjoji
M
M
Tiebel
M
M
Ochsner
S
S
Chan
L
L
Teng
B B
BB
D000256:Adenoviridae / Q000235:genetics; D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015316:Genetic Therapy; D006801:Humans; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D016337:RNA, Catalytic; D012333:RNA, Messenger