Spacer-length dependence of programmed -1 or -2 ribosomal frameshifting on a U(6)A heptamer supports a role for messenger RNA (mRNA) tension in frameshifting

Lin, Zhaoru; Gilbert, Robert J. C.; Brierley, Ian

The reduction in +1/À2 FS frequency seen with the U 4 UCU heptamer ( Figure 3 ) was thought-provoking in that it raised doubts as to whether a 'traditional' +1 FS event was occurring. In this mRNA, the A-site base changes (UUA to UCU) would not preclude forward movement of the P-site tRNA decoding the zero-frame UUU codon onto the overlapping +1 frame UUU codon, yet production of the +1/À2 product was diminished. To rule out that this was an effect of the identity of the A-site tRNA (tRNA Leu versus tRNA Ser ) on+1/À2 FS, we revisited the experiment, but changing the first base of the U 6 A heptamer (to A, C or U) in the context of pFSHIV-AON stopAll to probe P-site tRNA re-pairing in the À1 frame. From previous studies (6, 12) , we expected that tandem À1 slippage of P-and A-site tRNAs would be compromised to a greater or lesser extent, since the P-site codon would be sub-optimal for repairing in most cases, whereas a +1 movement of the P-site tRNA, as outlined above, would be unaffected. However, as shown in Figure 4 , changing the first base to A, C or G effectively abolished the +1/À2 product. The effect on À1 FS was consistent with earlier work, with a reduction in all cases, especially with C at the first position. These data suggested strongly that the +1/ À2 FS product results from À2 FS and this was confirmed by MS (for convenience, these data are presented at the end of the 'Results' section, since acquisition of sufficient trans-frame protein for MS analysis required additional knowledge obtained from experiments outlined in the following sections). The stimulation of both À1 and À2 FS on the U 6 A heptamer could also be engendered by an RNA-only oligonucleotide (15RNA) and a shorter 2-O-Me oligonucleotide (15OMe) (Supplementary Figure S1 ). Thus the capacity to stimulate both frameshift events is not specific to 25OMe. To confirm that AON-mediated À1 and À2 FS could also take place in a cellular context, the key elements of pFSHIV-AON were cloned into the dual luciferase frameshift reporter plasmid p2luc (33) such that the downstream firefly luciferase gene reported either À1 (p2lucHIV-AON À1 FS) or À2 (p2lucHIV-AON À2 FS) frameshifting, with the spacer length optimized in each case (À1 FS, 3 nt; À2 FS, 2 nt; see below). The relevant reporter plasmid together with increasing concentrations of 25OMe were transfected into COS 7 cells and luciferase activities measured 24 h later. Both À1 and À2 FS were detectable, with peak values of 2.6% (À1 FS) and 1.5% (À2 FS) and saturation at around $100 nM AON ( Figure 5) . A version of the À1 FS reporter plasmid with the IBV slippery sequence (p2lucIBV-AON) was also Spacer-length dependence of programmed À1 or À2 ribosomal frameshifting on a U 6 A heptamer supports a role for mRNA tension in frameshifting Based on the published literature, including our own studies of 80S ribosomes stalled at the IBV frameshift-stimulatory pseudoknot (22, 37) , we proposed a mechanical model of frameshifting in which a failure of intrinsic ribosomal helicase activity (15, 28) to unwind efficiently the stimulatory RNA during the translocation step leads to the build up of tension in the mRNA and subsequently, breakage of codon:anticodon contacts and realignment of the tRNAs in the À1 reading frame. The validity of this model remains to be determined, but one prediction of it is that the magnitude of frameshifting should be influenced by relatively subtle changes to the length of the spacer separating th