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

In recent years it has been discovered that efficient À1 FS can also be stimulated in some circumstances simply by annealing an RNA oligonucleotide downstream of a slippery sequence, at least in vitro (30) (31) (32) . This was unexpected as mRNA-antisense oligonucleotide (AON) complexes appear to lack the structural features of naturally occurring stimulatory RNAs, such as stem-stem junctions, base triplexes or kinks, that have been associated with models implicating resistance to unwinding (reviewed in 3, 4) . In an attempt to gain insight into the mechanism of AON-induced À1 FS, we initiated a study to examine the effect on À1 FS of modulating the spacing distance between slippery sequence and annealed AON. During the initial in vitro translations carried out to validate the system, we were intrigued to observe 'two' frameshift products in the AON-stimulated frameshift assays. In this article, we describe our examination of the origin of these products. We show that in the experimental system employed, based on that developed by Howard and colleagues (30) , both À1 'and' À2 FS can occur efficiently on the HIV-1 slippery sequence (U 6 A) in response to bound AONs. Importantly, this effect is also seen when the AON stimulator is replaced by a stem-loop or pseudoknot stimulator. By examining À1 and À2 FS outcomes on mRNAs with varying slippery sequence-stimulatory RNA spacing distances, we found that the spacer-length optimum for maximal frameshifting is different depending upon the kind of stimulatory RNA employed, and that À2 FS is optimal at a spacer length 1-2 nt shorter than that optimal for À1 FS. We propose that the shorter spacer increases the tension on the mRNA such that when the tRNA detaches, it more readily enters the À2 frame on the U 6 A heptamer. These experiments provide the first observation of À2 FS on a eukaryotic viral heptameric slippery sequence and support the view that mRNA tension is central to the mechanism of frameshifting, not only with 'traditional' stemloop or pseudoknot RNAs, but also with AON stimulators.