Variables affecting antisense RNA inhibition of gene expression.
We have sought to determine what variables affect the extent of inhibition of gene expression by stable nuclear-derived antisense RNAs. Myosin heavy chain gene II (MHCII) expression in Dictyostelium was used as a model system, because previous results have shown that nearly complete inhibition of expression can be achieved under appropriate conditions. Various fragments of the myosin gene were inserted into several transformation vectors in both sense and antisense orientation, and the effects on expression of protein and RNA from the endogenous MHC II gene were assayed. The results indicate that the critical factor was the particular fragment of the gene used to produce the antisense RNA. Some fragments produced complete inhibition of expression, whereas others gave only slight inhibition. The fragments that produced the greatest inhibition were from the tail region of the gene. In addition, cells were capable of overcoming the inhibition while still expressing the antisense RNA. We hypothesize that the three-dimensional topology of the antisense and sense RNAs determines their accessibility for interstrand hybridization. Transformation with sense fragments of the myosin gene also caused inhibition of the endogenous myosin gene. The inhibition seen with sense expression is not as dramatic as that for antisense, but it had the same phenotypic consequences for the cells. This phenomenon, which has now been documented in other systems, may be mechanistically similar to inhibition by antisense RNA in our system.
Scherczinger
C A
CA
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269.
Yates
A A
AA
Knecht
D A
DA
D000818:Animals; D004023:Dictyostelium / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects*; D017125:Genes, Protozoan / Q000235:genetics*; D005822:Genetic Vectors; D009218:Myosins / Q000235:genetics*; D016372:RNA, Antisense / Q000494:pharmacology*; D014170:Transformation, Genetic
D016372:RNA, Antisense; D009218:Myosins