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

Our experimental system for studying AON-mediated frameshifting in vitro is based on that of Howard et al. (30) and is shown in Figure 1 . We began by confirming that a 25 nt long morpholino (25MO) or 2-O-Me (25OMe) AON could stimulate-1 FS at the slippery sequence of HIV-1 (U 6 A) when bound 3 nt downstream on the mRNA. The frameshift reporter mRNA was transcribed from Nco I-cut pFSHIV-AON, a derivative of pFScass 5 (12; see section 'Materials and Methods'), and translated in rabbit RRL in the presence of increasing concentrations of AON. As can be seen in Figure 1 , both non-frameshifted (stop, 18 kDa) and À1 FS product (28 kDa) were seen, with the À1 FS efficiency peaking at $30%, a level similar to that measured for a control, pseudoknot-dependent frameshift signal (pFScass 5 IBV PK; 44% in this experiment). In the absence of added AON, the baseline À1 FS efficiency was 4-6%, indicating that the U 6 A heptamer is inherently slippery in RRL, as observed previously (12, 30) . In control translations with a non-targeting AON, or of mRNAs with mismatches in the AON binding site, very little frameshifting was seen, confirming the specificity of AON-mediated frameshifting (data not shown). Also evident in the 25OMe titration of Figure 1 was an unexpected product migrating just below the stop product. Based on the nucleotide sequence of the frameshift region and the position of termination codons in the different reading frames, this protein may correspond to ribosomes that have undergone a +1 (or À2) frameshift on the U 6 A heptamer. Alternatively, it could represent a peptide derived from ribosomes that had irreversibly stalled at the annealed AON, and subsequently dropped off the template (drop-off or d.o.). The unexpected product was also present, albeit to a lesser extent, in the 25MO titration. Given the possibility that it may represent an alternative frameshift product, we examined whether its generation was linked to the homopolymeric nature of the U 6 A slippery sequence by changing the slippery sequence of pFSHIV-AON to that present at the IBV (UUUAAAC) or simian retrovirus 1 (SRV-1) gag/pro (GGGAAAC) frameshift sites ( Figure 2 ). With these mRNAs, little or no +1/À2/d.o. product was evident, indicating that its appearance is most likely linked to the U 6 A slippery sequence rather than to a compromise of elongation arising from the presence of a stably bound AON. Examining the AON titrations further (Figures 1 and 2) , it can be seen that the plateau of À1 FS stimulation with 25MO was reached slightly earlier (at about 1 mM) than for 25OMe (3-5 mM). However, 25OMe was the more effective stimulator, promoting 49% À1 FS on UUUAAAC (c.f. 22% with 25MO), 42% on GGGAAAC (19% with 25MO) and, on the assumption that the novel product seen with U 6 A is an alternative frameshift product, a total of 74% frameshifting on U 6 A (33% with 25MO). The À1 FS efficiency engendered by 25OMe on the IBV and SRV-1 gag/pro slippery sequences (49% and 42%, respectively) was greater than that seen with that of HIV-1 (27%). However, it is likely that À1 FS on U 6 A would be greater if a proportion of ribosomes were not entering another frame.