Antisense-induced ribosomal frameshifting

Henderson, Clark M.; Anderson, Christine B.; Howard, Michael T.

The ability of cis-acting RNA structures or trans-acting 2 0 -O-Methyl antisense oligonucleotides to induce ribosomal frameshifting was determined by in vitro transcription and translation of a dual luciferase reporter vector, p2Luc. p2Luc contains the Renilla and firefly luciferase genes on either side of a multiple cloning site, and can be transcribed using the T7 promoter located upstream of the Renilla luciferase gene (45) . Sequences containing shift-prone sites were cloned between the two reporter genes such that the downstream firefly luciferase gene is in the +1 reading frame. The resulting constructs were then transcribed and translated in vitro with or without complementary cis-Acting stimulators of frameshifting at the antizyme shift site Initially, three dual luciferase reporter vectors were generated containing the human antizyme 1 frameshift cassette (p2luc-AZ1wt) with the 5 0 and 3 0 stimulators of frameshifting, with the pseudoknot deleted (p2luc-AZ1PKdel), or replaced with a stem-loop (p2luc-AZ1hp) (Figure 1 ). Each constructs was then subjected to coupled transcription and translation reactions in the presence of increasing amounts of spermidine, and the 35 S-labeled products separated by SDS-PAGE. Table 2 ). Maximal levels of frameshifting were found to occur when 2-4 mM of antisense oligonucleotide was added to the transcription/translation reactions (Supplementary Table 3 ). In the presence of 0.4 mM exogenous spermidine, highly efficient shifting of ribosomes into the +1 reading frame (higher than that observed in the wild-type antizyme frameshift cassette) was observed with the addition of AZ1A (26.1%), AZ1B (51.8%) and AZ1C (31.8%) (Supplementary Table 2 ). The most efficient frameshifting is observed with the antisense oligonucleotide AZ1B which anneals such that spacing between the shift site and the beginning of the duplex region is the same as that observed between the shift site and the beginning of stem 1 of the natural antizyme 3 0 pseudoknot structure (i.e. each has a 3 nt spacer). To verify that the antisense oligonucleotide was activating ribosomal frameshifting and not transcription slippage, RNA was transcribed from p2luc-AZ1PKdel in the absence of oligonucleotide and added to reticulocyte lysate translations in the presence of increasing amounts of 2 0 -O-Methyl AZ1B oligonucleotide. Frameshifting levels were increased to the same level as that observed in coupled transcription and translation reactions demonstrating that the oligonucleotide acts to induce frameshifting during translation (Supplementary Figure) .