The frequencies of translational misincorporation and stop codon readthrough are in the range of 10−3
, while more deleterious frameshifting errors occur even less often (10−5
). Although very low levels of translation errors can be detected in vivo
, the signal/noise ratio of such quantification is low and can mask significant variations, especially decreases in error rate. Programmed translational errors include several related phenomena, known collectively as recoding, which end with a local alteration of the normal rules of decoding (32
). The mechanisms at play generally involve only normal components of the translation machinery, and a number of studies have demonstrated the role of both mRNA sequences and higher structures in stimulating errors. These sequences provide valuable tools to monitor the efficiency of translational accuracy in various conditions, and significant variations in programmed errors can easily be quantified using reporter constructs (31
). Here, we used a dual reporter system that relies on the relative levels of luciferase versus β-Galactosidase activities when encoded in the same mRNA, but separated by a sequence that induces a high error level. Accuracy of the elongation step of translation was systematically examined here using sequences known to promote either −1 (IBV) or +1 (EST3) frameshifting. For the termination step, a readthrough sequence derived from the TMV that contains the UAG stop codon in-frame was used. When required, the +1 OAZ frameshifting sequence and the UAA and UGA stop codons in the TMV context were tested (see ‘Materials and Methods’ section).
The mutants examined for each ribosome region are depleted of one or more modifications, caused by genetic deletion of the corresponding guide snoRNAs. The modifications affected are described in and . The test strains for the three regions (ASF, DC and H69) have been described previously. Results from these studies have been integrated in the tables. Error rates (readthrough and frameshifting) displayed by each mutant strain were compared to those obtained in the WT, and a mutant/WT factor was determined. At least five independent experiments (up to 20) were performed. The significance of variation was evaluated using a non-parametric statistical test to calculate a P-value (Mann–Whitney).
Figure 1. Modification map of S. cerevisiae rRNA. (A) Secondary structure of the small subunit (SSU) and large subunit (LSU) rRNAs with Nm and Ψ modifications. The modification-rich regions featured in this study are boxed. (B) Modifications in the decoding (more ...)
Modifications in the three functional regions examined
The ASF is formed by a long helix (Helix 38 or H38) in folding domain II of the 25 S rRNA, and is conserved in all three kingdoms of life. The ASF is part of the B1a intersubunit bridge between the large and small subunits. It protrudes into the intersubunit space above the tRNA at the ribosomal A site and makes contact with the A-site tRNA through the tip region of H38, and with 5 S rRNA through a segment in the upper portion of the ASF. In E. coli the ASF also establishes contact with protein S13 (S15 for Saccharomyces cerevisiae). H38 in S. cerevisiae contains seven Ψs and three other Ψs occur in short helices that flank H38. The neighboring helices (H37 and H39) fold together with the basal portion of H38 and may be in position to influence the behavior of the ASF.
A total of six modifications were analyzed in the ASF region, four in the middle portion of H38 and two from the flanking helices (C). We note that the bacterial ASF lacks modifications, whereas eukaryotic ASF helices have several Ψs and three of these are conserved in mammals as well as yeast. The deletions examined here include the cluster of these conserved modifications (). Three Ψs in the ASF were not included as the corresponding snoRNAs also direct modifications to three other rRNA positions outside of the ASF, which would complicate data interpretation. Thus, the mutants studied only lack Ψs in the ASF region itself or in combination with one or two Ψs in the adjacent helices. There were five mutants deleted variously for one, two and all six modifications.
No significant effects were observed with mutants deleted of one or two of the six Ψs, on readthrough (UAG stop codon) and −1 frameshifting levels (, columns 2–5). Small effects were seen for +1 frameshifting for two mutants: one lacking a single modification (Ψ1042; , column 2), and the other lacking two modifications (Ψ960 and Ψ986; , column 5); here, slight, but significant decreases in error rate were detected (0.8-fold WT). These results indicate that none of the six ASF modifications tested has a strong effect on translation accuracy. A strain deleted of all six modifications exhibited no change in the capacity to maintain correct reading frame (+1 and −1) or to recognize the UAG stop codon (, column 6). In previous studies, mutations in the distal portion of the ASF were shown to affect the accuracy of translation. Shortening the E. coli
ASF caused an increase (~1.44-fold) of +1 frameshifting (36
) while in S. cerevisiae
, a point mutation led to a decrease (~0.65-fold) in suppression of a UAA nonsense codon (37
). These data prompted us to test the readthrough level using UGA and UAA targets with the multiply depleted test strain; UGA is the least efficient stop codon in eukaryotes. A significant increase in UGA suppression level was revealed (1.4-fold, P
< 0.037). In contrast, readthrough of the UAA stop codon was equivalent to the WT level (P
= 0.148). We also tested a sequence derived from the S. cerevisiae OAZ
gene, which drives +1 frameshifting. A UGA termination codon is part of the conserved OAZ
frameshift site and is the main stimulating element in S. cerevisiae
). The frameshift level was affected, showing a significant decrease (0.7-fold, P
< 0.0001). This result confirms the protective effect of this Ψ cluster on UGA readthrough by an independent way.
Translational accuracy of A-site finger mutants
In summary, loss of individual modifications in the ASF and flanking helices slightly modifies the maintenance of translational reading frame. No effect is observed when using a combination of all six Ψ deletions. This situation causes a new phenotype, namely, elevation of UGA nonsense codon readthrough that is correlated with an impact on the OAZ +1 frameshift level.
The DC, located in the ribosomal small subunit, includes the A and P sites of codon–anticodon interactions and thus, has major influences on translation accuracy. It was therefore of great interest to test the functional relevance of rRNA modifications in this region. Data from structural studies, chemical protection, cross-linking and genetic analysis in E. coli
suggest that the DC includes portions of helices 24, 31, 34, 44 and the 530 loop of 16 S rRNA. The homologous regions in yeast are presumed to be functionally equivalent. In E. coli
, the G530 loop and A1492 and A1493 of 16 S rRNA play a central role in control of codon–anticodon pairing accuracy, through the proofreading activity of the ribosome (39
). The functional DC is not fully delimited and includes rRNA segments that are separated in the primary structure, but are close to one another in crystal structures. The DC region in yeast contains eight modified nucleotides: five Nm and three Ψ; one Ψ undergoes additional modifications to form 1-methyl-3-(3-amino-3-carboxypropyl) Ψ(m1
Ψ) (B). Seven of these modifications occur in mammalian cells and two occur in E. coli
(). In yeast, four of the eight DC modifications are in the A-region. Two are located on the tRNA-entry side of the A-site tRNA and we will refer to these as A-region modifications (Um578 and Gm1271). The other two are located above tRNA in the A site and we will refer to these as Aa-region modifications (A
bove) (Ψ1187 and Gm1428). Of the four remaining modifications, two are in the P-region (m1
Ψ1191 and Cm1639), and two are in the E-region (Ψ999 and Cm1007).
Analyses of translation accuracy were carried out using a set of 14 deletion mutants, lacking individual modifications (four mutants) or different combinations of modifications (10 mutants) () (30
). Two strains that lack modifications in the P-region or a combination of the Aa- and P-regions exhibited a significant change in termination accuracy, which was readily apparent using the UAG stop codon (1.3–1.5-fold of WT level; , columns 3 and 9). Both mutants lack Cm1639 in the P-region: one lacks this Nm alone, the other also lacks m1
Ψ1191 (P-region) and Gm1428 (Aa-region). Interestingly, three other combinations of Cm1639 deletions did not affect termination, including combinations with Gm1428 or with Ψ1187 and m1
Ψ1191, all in the Aa- and P-regions (, columns 8, 14 and 15). This striking observation indicates that the effect depends not only on a particular modification, but also on the specific pattern of deletions and residual modifications, suggesting strong synergistic relationships between modifications. Altogether, the deletion results show that translation termination has low sensitivity to loss of modifications in the DC region.
Translational accuracy of decoding region mutants
In contrast, moderate to strong frameshifting effects were detected for 11 of the 14 strains examined (for either +1 or −1 contexts). As for readthrough, deleting the P-region Cm1639 had an impact on +1 frameshifting alone or in four combinations with other Aa- and P-region deletions. The +1 frameshift activity was 1.7-fold that of WT for loss of the Cm1639 modification alone (, column 3) and varied from 1.8- to 2.4-fold for the other mutant strains, which included two, three and four deletions from the Aa- and P-regions (; columns 8, 9, 14 and 15). Blocking the two E-region modifications reduced +1 frameshift activity (0.6-fold; , column 6). The values for four other mutants with three deletions each (, columns 10–13) are not statistically different from those obtained with the various single deletions (P-values ranging from 0.244 to 0.579). These last strains included two mutants deleted of both A-region modifications and one Aa-region modification; one mutant deleted of one A-region, one Aa-region and one P-region modifications; and one mutant deleted of two modifications in the Aa-region and one modification in the P-region.
On examining effects on −1 frameshifting, we observed that loss of three individual modifications alone from the Aa- and P-regions increased frameshift activity (Ψ1187; , column 5, Gm1428 column 2 and Cm1639 column 3, respectively). Notably, loss of Ψ1187 from the Aa-region led to a 1.2-fold increase in −1 frameshifting, but to a decrease when combined with deletion of a P-region modification (m1acp3Ψ1191; , column 7) or with two A-region modifications (Um578 and Gm1271; , column 10) (0.5-fold). Increased −1 frameshift activity was also observed for two strains deleted of a mix of Aa- and P-region modifications, as follows: one Aa- and one P-region modification (, column 8), one Aa- and two P-region modifications (, column 9). No effect was detected for a strain deleted of the two E-region modifications. Similarly, no effect was detected for four multiply deleted strains lacking other combinations of A-, Aa- and P-region modifications (, columns 11–14). Behavior of one strain lacking four modifications was difficult to interpret due to internal variations of error level that resulted in a P-value of 0.110 (, column 15).
The rRNA site that undergoes hypermodification in yeast (m1
Ψ1191) is also modified at the corresponding site in E. coli
, yielding mG966 in the 970 loop of 16 S rRNA. Interestingly, the 970 loop is involved in tetracycline binding (40
) and a mutation at position 966 confers resistance to the antibiotic. These relationships prompted us to test the sensitivity of the m1
Ψ1191 deletion strain to doxycycline, a tetracycline analog. Strikingly, this strain showed substantially increased resistance to doxycycline. This finding suggests that tetracycline and related analogs target the same position in both organisms (A).
Figure 2. Sensitivity of rRNA modification mutants to translational inhibitors. WT and mutant strains lacking individual modification were spotted as 10-fold dilutions on YPD and YPD containing the indicated antibiotic media. Cells were incubated for 3 days at (more ...)
In summary, modifications in the Aa-, P- and E-regions have significant roles in maintaining translation accuracy, whereas the two A-region modifications have only minor influence. Both Aa-region and P-region modifications are involved, as blocking these individually affects either stop codon readthrough or +1 and −1 frameshifting. Fidelity is also altered with deletion of multiple Aa- and P- region modifications in different combinations. Effects on both readthrough and +1/−1 frameshifting were seen for two strains deleted of at least the P-region modification Cm1639. Finally, blocking the two E-region modifications together only affects +1 frameshift activity. Taken together, the data indicate that Aa- and P-region modifications have greater influence on maintaining normal accuracy than A-region modifications.
H69 is a short stem–loop structure of 19 nucleotides located in domain IV of 25 S rRNA. It is a component of the intersubunit bridge B2a, along with portions of helix 44 of 18 S rRNA. In addition to its important role in subunit joining, it has been established in E. coli
that the H69 loop and stem contact the D-stem of tRNAs in the A site and P site, respectively, suggesting a role(s) in translation accuracy. Interactions between H69 and various translational factors have also been described for E. coli
. In particular, relationships between H69 and RF2 and RF3 were established by cryoelectron microscopy experiments, linking H69 with termination of protein synthesis (42–44
). Earlier evidence of these relationships came from the observation that blocking formation of E. coli
H69 modifications Ψ1911, Ψ1915 and Ψ1917, by inactivating the pseudouridine synthase RluD
, impaired translation termination; readthrough activity increased by up to 14-fold for the UGA codon (6
). Further evidence of the importance of H69 comes from our findings that yeast lacking assorted H69 modifications can be defective in growth, subunit joining and translation rate (5
). Mutants from that collection are featured in the present study.
The relatively small H69 domain in S. cerevisiae
is rich in modifications, with two Ψs in the stem (Ψ2264, Ψ2266) and two Ψs plus one Nm in the loop (Am2256, Ψ2258, Ψ2260) (D). All these modifications are conserved in humans. The Ψ2258 modification (Ψ1915 in E. coli
), is of particular interest evolutionarily, since it is conserved in bacterial variants with the minimal genomes described thus far (45
). These five modifications are guided by four snoRNAs, with one snoRNA targeting two sites (). We tested six strains deleted for individual modifications and in three combinations of two to four deletions.
Remarkably, the results point to the involvement of at least four and possibly all five modifications in translation accuracy for both the elongation and termination phases (). Readthrough was affected for each stop codon tested with the four single deletion strains. The general trend was a decrease of readthrough (between 0.3- and 0.7-fold). The mutant lacking Ψ2258 and Ψ2260 (, column 4) was an exception, showing a small increase for the UAG stop codon (1.2-fold). Frameshift efficiency was affected to a small, but significant extent (0.4- to 0.7-fold WT) for three mutants lacking one modification and the mutants lacking two to three modifications. Two strains were affected in both +1 and −1 frameshifting (lacking either Ψ2266 alone or lacking Ψ2258 and Ψ2260; , columns 3 and 4), but strains lacking Ψ2264 or Am2256 alone (, columns 5 and 2) displayed a normal level of +1 or −1 frameshifting, respectively. Strikingly, loss of Ψ2266 or both Ψ2258 and Ψ2260 altered nonsense suppression and reading frame maintenance, both forward and backward.
Translational accuracy of Helix 69 mutants
For strains lacking three or four modifications (, columns 6 and 7), a small decrease in accuracy was observed for both readthrough and frameshifting, although the differences were not statistically significant. For stop codon recognition and −1 frameshifting, strains deleted of three or four modifications behaved similarly to WT controls. However, the strain deleted of three modifications displayed a small decrease of +1 frameshift levels (0.8-fold WT) (). We did not test a strain deleted for all five modifications, due to a strong growth impairment that did not allow precise quantification of translation accuracy (5
Sensitivity to aminoglycoside antibiotics was evaluated as well. These drugs interfere with translocation of tRNA from the A site to the P site and induce translational misreading. Three mutants lacking one or two modifications show increased resistance to either one or two of the three drugs, in strain-specific fashion (B). Deleting Ψ2264 (in the stem) caused increased resistance to kanamycin, but not to G418 or neomycin, whereas deleting Ψ2258 and Ψ2260 (both in the loop) yielded increased resistance to G418 and kanamycin, but not to neomycin. The differences presumably reflect different binding patterns for the drugs, as evidenced by different positional effects of modification on binding.
In summary, three single deletions and one double deletion of H69 modifications altered both elongation and termination accuracy. Interestingly, loss of three or four modifications has only a slight effect, consistent with some of the combinatorial effects observed for the DC and ASF. Three mutants showing increased translation accuracy also exhibit increased resistance to aminoglycoside antibiotics, suggesting a correlation between translation fidelity and ribosome structural changes.