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 situation is even more convoluted when one considers AON-mediated frameshifting (30) (31) (32) 50) . The mRNA-oligonucleotide complexes would appear to lack unusual features like stem-stem junctions, triplexes or kinks and thus the mechanism by which they induce frameshifting is uncertain. It is known that the length of the AON can affect the efficiency of the process (32), thus the stability of the mRNA:AON duplex plays a role. However, while the specific chemical modifications present in 2-O-Me, MO, phosphorothioate (30) and locked nucleic acid (32) AONs can affect binding stability and target specificity, they are not fundamental for activity in frameshifting, as unmodified RNA oligonucleotides can also stimulate À1 FS, at least in vitro (30, 31) and also À2 FS (Supplementary Figure S1) . Recently, it has been shown that stem-loop structures can effectively substitute for RNA pseudoknots in some circumstances, with frameshift-stimulatory activity driven largely by the thermodynamic stability of the stem, but also influenced by loop size, composition and stem irregularities (51) . The stem-loop employed here is also clearly capable of inducing frameshifting at certain spacer distances (Figure 7) . With this in mind, it is likely the AONmediated frameshifting is largely determined by stability and duplex length (32) . Nevertheless, the encounter between ribosome and AON is clearly different from that of a cis-acting secondary structure. Firstly, the 80S helicase will encounter the 3 0 -hydroxyl of the annealed AON rather than a constrained duplex (or triplex in some pseudoknots). Secondly, the optimal spacing for frameshifting is quite different, being only 3 nt for AON-mediated À1 FS, but respectively, some 7 nt and 8 nt for optimal stem-loop or pseudoknot-mediated À1 FS. The simplest interpretation of these observations is that the putative 80S helicase unwinds several base-pairs of the annealed AON before its activity is compromised. Further work will be required to confirm a role for the helicase in frameshifting and to understand how it is compromised by what appears to be a regular mRNA:AON duplex. It may be that the presence of the free 3 0 -end of the AON is critical in this regard.