This work describes a comprehensive analysis of the S.cerevisiae genome which attempts to identify cellular recoding events occurring during translational termination. We have developed a genomic approach, seeking genes with an extended coding potential, through the stop codon, without prior bias from existing ideas on termination codon suppression mechanism. The candidate genes are composed of two ORFs separated by a unique stop codon named SORF. We fixed the minimal length of the second ORF (ORF2) at 200 nt, and only SORFs without an ATG codon in the first 50 nt after the stop codon were retained for analysis. This allowed us to analyze a reasonable number of candidates, and excluded from the analysis genes possibly expressed through internal ribosome entry or translational reinitiation. Our preliminary data suggest that such genes, controlled through translation initiation, could also be retrieved by this kind of approach (O.Namy, I.Hatin and J.P.Rousset, unpublished results).
Recently, Harrison et al
. have published an analysis of the yeast genome seeking ‘disabled ORF’, calling these dORF or mORF (39
). Their results overlap only slightly with ours, because we did not base our analysis on homologies with known genes, and limit our research of 3′ extensions to official ORFs. They identified 11 of the SORFs characterized here (YDR082W, YIR044C, YER039C-a, YHR058C, YKL031W, YKL020C, YLR465C, YMR057C, YNR069C, YOR024W, YOR051C), two of which (YLR465C and YNR069C) display a significant stop codon bypass efficiency.
Our approach identified 58 SORFs in the yeast genome. Eight sequences displayed a stop codon bypass efficiency 10-fold higher than background. For each of these candidates, a unique mRNA covering both ORFs is present in the cell. Although it is only for those SORFs showing the highest bypass levels that one could expect to detect an mRNA editing mechanism, we did sequence the RT–PCR products for each SORF. No RNA post-transcriptional modification was identified. Moreover, from the amplification of the mRNA using a poly(dT) primer at the reverse transcription step, we concluded that these mRNA are polyadenylated and not rapidly degraded.
We quantified the stop codon bypass efficiency for each of the eight BSC genes in two isogenic [PSI+] and [psi–] yeast strains. In the [PSI+] strain, we expected that if the bypass mechanism is bona fide readthrough, the stop codon efficiency should increase. In fact, we observed a stop codon bypass increase only for the genes BSC4 and IMP3. For the other sequences, no such difference between the [psi–] and the [PSI+] strains was observed; thus for these sequences, the termination process is less dependent on the concentration of the eRF3 release factor, or the stop codon is bypassed by another translational mechanism independent of eRF3. The discrepancy between the results obtained with both wild-type [psi–] strains (Y349 and 74-D694) is not clear. In any case, these results indicate that the genetic background influences these unknown stop codon bypass mechanisms.
Recoding signals are usually composed of two elements, the sequence where the recoding takes place, and a stimulatory sequence. In a few examples, the stimulatory sequence is simply the immediate 3′ stop codon nucleotide context (26
). However, most frequently the stimulatory sequence is a pseudoknot (or a stem–loop), and may be as far as 4 kb from the recoding site (40
). A computational analysis of the stop codon 3′ nucleotide context of the BSC
genes did not reveal any significant secondary structure in the vicinity of the stop codon. However, the BSC1
sequences displayed an unusual sequence pattern. The BSC1
sequence included four repeats of a nine amino acid motif that correspond to imperfect nucleotide repeats. This suggests that the role of these motifs is at the level of the protein and not of the mRNA. The exact role of these motifs in protein function is still unknown; however, our results indicate that this region is essential to obtain a high level of stop codon bypass, which suggests that the nucleotide motif is involved in the bypass mechanism. It is interesting to note that another recoding event, hopping, uses a codon repetition to promote stop codon bypassing (25
). One can thus speculate that the mechanism active in BSC1
would not be readthrough, but hopping. This could explain the lack of effect of the PSI factor on the stop codon bypass efficiency. More experiments are necessary to elucidate the precise mechanism.
The study of BSC4 indicated that the stop codon is present in a typical readthrough context. This observation is coherent with the increase of the stop codon bypass efficiency observed in the [PSI+] strain. Our results demonstrate that the immediate stop codon context is in fact sufficient to promote a high level readthrough of the BSC4 stop codon.
Overall, our results emphasize that recoding events take place at the stop codon more often than expected. These events should be carefully sought during genome annotation as shown recently in euplotes (20
). They also suggest that mechanisms other than readthrough are possibly used by cells to allow ribosomes to bypass the stop codon.