Antisense-induced ribosomal frameshifting

Abstract

Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1-ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem-loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5' of the shift site and to polyamine levels.

Figure 1

(A) Reporter construct design: cis- and trans-acting stimulators of frameshifting. Sequence of the shift site and downstream sequences for dual luciferase constructs containing cis-acting structures used in this paper. P2luc-AZ1wt contains the wild-type antizyme frameshift cassette, p2luc-AZ1-0sp has a 3 bp deletion of the spacer sequences separating the shift site from the pseudoknot and p2luc-AZ1hp contains a hairpin replacement of the pseudoknot structure. S1 and S2 refer to stem 1 and stem 2 of the RNA pseudoknot. L1 and L2 refer to loops 1 and 2 of the pseudoknot. Fluc and Rluc represent Firefly and Renilla luciferase genes, respectively. (B) Sequence of the shift site and downstream sequences for dual luciferase constructs and their complementary antisense oligonucleotide partners. Fluc and Rluc represent Firefly and Renilla luciferase genes, respectively.

Figure 2

Cis-acting 3′ stimulators of frameshifting. Plasmids p2Luc-AZ1wt (AZ1wt), p2luc-AZ1PKdel (AZ1PKdel) and p2luc-AZ1hp (AZ1hp) were transcribed and translated in rabbit reticulocyte lysate in the absence or presence of increasing amounts of spermidine (final concentration indicated in mM). SDS–PAGE of ³⁵S-methionine-labeled protein products from transcription and translation reactions is shown. The location of the full-length frameshift product (+1 FS) and non-frameshift termination product (Term) are indicated. The efficiency of frameshifting (% FS) is indicated.

Figure 3

Antisense oligonucleotide stimulators of frameshifting. Plasmids p2Luc-AZ1PKdel (AZ1PKdel) or p2luc-AZ1-0sp (AZ1-0sp) were transcribed and translated in rabbit reticulocyte lysate in the absence or presence of increasing amounts of spermidine (final concentration indicated in mM). Either 2′-O-Methyl antisense oligonucleotide AZ1A (A), AZ1B (B) or AZ1C (C) was added to the transcription and translation reactions at 2 μM final concentration. No antisense oligonucleotide was added to reactions with AZ1-0sp (D). SDS–PAGE of ³⁵S-methionine-labeled protein products from transcription and translation reactions is shown. The efficiency of frameshifting (% FS) is indicated.

Figure 4

Sequence effect 3′ of the shift site on 2′-O-Methyl induced frameshifting. Plasmids p2Luc-AZ1PKm1 (AZ1PKm1) (A and B) or p2luc-AZ1sl (AZ1SL) (C and D) were transcribed and translated in rabbit reticulocyte lysate in the presence of 0.4 mM spermidine. Either 2′-O-Methyl antisense oligonucleotide PKm1 (A), PKm2 (B), SL1 (C) or SL2 (D) was added to the transcription and translation reactions at increasing concentrations. Final concentration of the antisense oligonucleotide (AO) is indicated in μM. SDS–PAGE of ³⁵S-methionine-labeled protein products from transcription and translation reactions is shown. The efficiency of frameshifting (% FS) is indicated.

Figure 5

Sequence effect 5′ of the shift site on 2′-O-Methyl induced frameshifting. Plasmids p2Luc-AZ1FS (AZ1 FS) (A and B) or p2luc-AZ1FS-UGG (AZ1FS-UGG) (C and D) were transcribed and translated in rabbit reticulocyte lysate in the presence or absence of 0.4 mM spermidine. Either 2′-O-Methyl antisense oligonucleotide AZ1A, AZ1B or AZ1C were present. SDS–PAGE of ³⁵S-methionine-labeled protein products from transcription and translation reactions is shown for AZ1FS (A) and AZ1FS-UGG (C). The efficiency of frameshifting (% FS) is indicated.

Figure 6

Antisense induced frameshifting in cultured mammalian cells. Plasmid p2lucAZPKdel was transfected into cultured CV-1 cells along with increasing amounts of 2′-O-Methyl antisense oligonucleotide AZ1B. The cells were incubated for 20 h and the percent frameshifting was determined by assaying firefly and Renilla luciferase activity in cell lysates as described in Materials and Methods.