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 an attempt to disentagle possible origins of the +1/ À2/d.o. product, a variant of pFSHIV-AON was prepared (pFSHIV-AON stopAll) in which three local stop codons in the 0 and +1 reading frames were changed to sense codons such that the length of the ORFs downstream of the U 6 A stretch (in each of the three frames) would allow differentiation of a product of +1/À2 FS from that of a d.o. event based on size ( Figure 3 ). This experiment revealed that the majority of the +1/À2/d.o. product appears to be derived from a +1/À2 FS, with a small proportion derived from a product whose size is consistent with prolonged (or permanent) stalling at the U 6 A heptamer (presumably) while the ribosome attempts to unwind the annealed oligonucleotide. Consistent with earlier work, with 25MO there was less +1/À2 FS product and little or no d.o. product, suggesting that morpholino oligonucleotides present less of a barrier to ribosomal elongation than their 2-O-Me counterparts. Within the context of pFSHIV-AON stopAll, three slippery sequence variants were prepared to address the question of whether 25OMe-stimulated +1/À2 FS was strictly dependent on a U-rich slippery sequence. As shown in Figure 3 , with slippery sequence U 4 UCU, possessing an A-site codon sub-optimal for frameshifting, both À1 FS and +1/À2 FS were diminished (3-to 4-fold) suggesting a requirement for optimal A-site tRNA re-pairing in each case (see below). With slippery sequence A 6 C, efficient À1 FS was observed (21%) but the quantity of +1/À2 FS product was reduced (albeit detectable at $4%). We also tested A 8 C and were surprised to see relatively high expression of both À1 and +1/À2 FS products in the absence of added AON. The synthesis of these products could potentially be accounted for by transcriptional slippage on A 8 C by SP6 RNA polymerase during synthesis of the reporter RNAs (35) . Alternatively, on this long homopolymeric A 8 C stretch, the ribosome can frequently lose frame even in the absence of a stimulatory element. A similar observation has been made for a U 8 stretch (12) . Significantly, in the presence of 25OMe, expression of the À1 FS product from the A 8 C mRNA rose to a level similar to that seen with the U 6 A mRNA and the +1/À2 FS product was also enhanced (to about half the value seen with U 6 A). Thus translational frameshifting is certainly taking place on this A-rich stretch and both À1 FS and+1/À2 FS products are synthesized.