Subsequently, we sought to confirm the interaction by investigating termination-reinitiation in yeast cells, exploiting the fact that the helix 26 equivalent in yeast 18S rRNA has a primary sequence distinct from that of the rabbit. Using a dicistronic reporter mRNA with variant motif 1 sequences, we found that increasing the complementarity of the motif to that of yeast 18S rRNA stimulated termination-reinitiation, supporting strongly the view that intermolecular interactions are important in reinitiation on the BM2 ORF. However, two aspects of this experiment require comment. First, a high background activity was observed in control assays (Figure 4) , the origin of which is uncertain Secondly, in yeast cells, the BM2wt reporter mRNA, whose motif 1 is not fully complementary to the helix 26 target, showed an efficiency of termination-reinitiation only two-fold lower than the BM2yeast mRNA. In RRL, a single base mismatch between mRNA and rRNA is sufficient to inhibit BM2 synthesis 10-20 fold [11] . One possible explanation for this difference is that the higher concentrations of mRNA and rRNA in an intact cell can act to stabilise the (presumably) weakened mRNA:rRNA interaction. During preparation of this manuscript, Luttermann and Meyers (2009) published a similar, more sophisticated, investigation of FCV motif 1:18S rRNA interactions using a yeast expression system in which both mRNA and rRNA partners could be The rlucM1/BM2fluc overlap window was changed from UAAUG to CAAUG such that termination occurs at the next in-frame stop codon, some 63 nt downstream of the original stop site (+63). New stop codons were then inserted, preserving the stop codon context, at +12, +24, +36 and +48 nt downstream of the natural termination site. Messenger RNAs derived from these plasmids and a control mRNA (ps) were translated and analysed as detailed in the legend to Figure 1 . This experiment is a repeat of that found in [11] and is shown here as a control. (B) The rlucM1/BM2fluc overlap window was changed from UAAUG to CAACU, and the UAAUG sequence reintroduced at +12, +24, +36, +48 or +63 nt downstream preserving both the initiation codon and stop codon context. Messenger RNAs derived from these plasmids were translated and analysed as above. doi:10.1371/journal.pone.0016822.g005 Figure 6 . Effect on termination-reinitiation of altering the BM2 start codon position. (A) The termination-reinitiation overlap was mutated from AUAAUG to AUAAGC and the BM2fluc start codon reintroduced at +12,+24, +36, +48 or +63 nt, preserving the start codon context at the -3 and +4 positions. The codon immediately 59 of the native reinitiation site is denoted -1, the reinitiation site is shown as +1. Messenger RNAs derived from these plasmids were translated and analysed as above. (B) Left panel: Translations were performed as in Figure 6A and the samples were separated by SDS-PAGE and autoradiographed. The putative AUA reinitiation product is marked with an asterisk. In addition, the three right hand modified. This work provided strong evidence in support of a requirement for mRNA:rRNA interactions in termination-reinitiation in the expression of FCV VP2 [16] .