Antisense RNA complementary to 3' coding and noncoding sequences of creatine kinase is a potent inhibitor of translation in vivo.

Abstract

Antisense RNA is a potentially powerful tool for creating dominant negative mutations, but one of the limitations of this strategy has been the relative inefficiency of antisense transcripts in blocking target gene expression. To identify more effective target sequences, helper-free retrovirusmediated gene transfer was used to introduce antisense RNAs complementary to multiple functional regions of the human creatine kinase B (CK-B) mRNA into U937 cells. Antisense RNA complementary to the last third of the coding and all of the noncoding region of this mRNA is highly effective; one or two antisense transcripts is sufficient to block the expression of one CK-B mRNA. In contrast, antisense RNA from which sequences complementary to the last 17 codons and all the 3' noncoding region have been deleted has no effect on CK-B expression. Neither antisense RNA alters the abundance of the target message, processing of the primary transcript, egress of the CK-B message from the nucleus, or the polysome profile of CK-B mRNA in sucrose gradients. These results point to a direct effect of the antisense transcript on translation and suggest that this effect may be explained at least in part by an inhibition of elongation or termination as a consequence of the duplex formed in the distal coding and/or 3' noncoding region.Antisense RNA has been used to block expression of target genes in prokaryotes and eukaryotes (1). General application of this strategy, however, has been limited by a lack of understanding of the basic rules for designing an efficient antisense system. Several systems suggest that the 5' noncoding region of the mRNA is a good target for antisense RNA (2, 3), while others report that coding sequences (4, 5), splice junction sites (6), and 3' processing sites (7) are also effective targets. Antisense RNA has been reported to block expression of the target gene by trapping of the duplex in the nucleus (8), accelerated turnover of the duplex leading to reduction in abundance of the target mRNA (3, 9-11), and altered processing of the target mRNA secondary to duplex formation (7, 12). Many reports (1, 3) indicate that the abundance of the antisense transcript must exceed that of the target mRNA by 100-fold or more to achieve even partial suppression. Efficient use of antisense RNA as a dominant negative mutation strategy would be enhanced if more effective mechanisms of antisense inhibition of gene expression were identified.The present study was undertaken to determine whether a more effective antisense strategy could be designed and, if so, to study the mechanism by which gene expression is blocked. Creatine kinase, an important enzyme in energy metabolism and an easily quantitated gene product, was selected as the target gene for these studies. Antisense RNA complementary to the area surrounding the stop codon was found to be a potent inhibitor of CK-B expression in U937 cells. Not only is antisense RNA complementary to this region of the CK-B mRNA potent; none of the previously described mechanisms of a...

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Prothymosin alpha antisense oligomers inhibit myeloma cell division.

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Regulation of lipoprotein lipase translation by epinephrine in 3T3-L1 cells. Importance of the 3' untranslated region.

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