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In Eubacteria, Unlike Eukaryotes, There Is No Evidence for Selection Favouring Fail-Safe 3' Additional Stop Codons

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In Eubacteria, Unlike Eukaryotes, There Is No Evidence for Selection Favouring Fail-Safe 3' Additional Stop Codons

Alexander T Ho et al. PLoS Genet.

Abstract

Errors throughout gene expression are likely deleterious, hence genomes are under selection to ameliorate their consequences. Additional stop codons (ASCs) are in-frame nonsense 'codons' downstream of the primary stop which may be read by translational machinery should the primary stop have been accidentally read through. Prior evidence in several eukaryotes suggests that ASCs are selected to prevent potentially-deleterious consequences of read-through. We extend this evidence showing that enrichment of ASCs is common but not universal for single cell eukaryotes. By contrast, there is limited evidence as to whether the same is true in other taxa. Here, we provide the first systematic test of the hypothesis that ASCs act as a fail-safe mechanism in eubacteria, a group with high read-through rates. Contra to the predictions of the hypothesis we find: there is paucity, not enrichment, of ASCs downstream; substitutions that degrade stops are more frequent in-frame than out-of-frame in 3' sequence; highly expressed genes are no more likely to have ASCs than lowly expressed genes; usage of the leakiest primary stop (TGA) in highly expressed genes does not predict ASC enrichment even compared to usage of non-leaky stops (TAA) in lowly expressed genes, beyond downstream codon +1. Any effect at the codon immediately proximal to the primary stop can be accounted for by a preference for a T/U residue immediately following the stop, although if anything, TT- and TC- starting codons are preferred. We conclude that there is no compelling evidence for ASC selection in eubacteria. This presents an unusual case in which the same error could be solved by the same mechanism in eukaryotes and prokaryotes but is not. We discuss two possible explanations: that, owing to the absence of nonsense mediated decay, bacteria may solve read-through via gene truncation and in eukaryotes certain prion states cause raised read-through rates.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Z-scores, measuring deviation in ASC frequency from a null model (10,000 simulations), plotted against the genomic GC3 content of filtered TT11 bacterial genomes.
A significant negative relationship is observed between Z-score and genomic GC3 across positions +1 to +6 (p < 2.2 x 10−16, ρ = -0.64, Spearman’s rank correlation).
Fig 2
Fig 2. ASC frequencies of TGA-terminating HEGs compared to TAA-terminating LEGs.
Each bar represents one genome, with bar heights representative of the standardised difference between the two groups. There is no significant difference between TGA-terminating HEGs and TAA-terminating LEGs at positions +2 to +6 (p = 0.060 for position +2, p = 1 for position +3, p = 0.83 for position +4, p = 0.60 for position +5, p = 0.62 for position +6, Wilcoxon signed-rank tests). There is prima facie significant difference between TGA-terminating HEGs and TAA-terminating LEGs at position +1 (p = 0.041, Wilcoxon signed-rank test), but this does not survive Bonferroni correction.
Fig 3
Fig 3. ASC frequencies calculated in TAA, TGA and TAG-terminating genes for all genes, highly expressed genes (HEGs) and lowly expressed genes (LEGs).
Error bars represent bootstrapped standard error. We find significant differences between primary stop groups at position +1 when considering all genes (p = 1.89 x 10−15, χ = 67.81, Kruskal-Wallis) and LEGs (p = 0.032, χ = 6.91, Kruskal-Wallis), but not HEGs (p = 0.14, χ = 3.97, Kruskal-Wallis). We instead observe significant enrichment at position +2 in HEGs (p = 0.029, χ = 7.09, Kruskal-Wallis). Signals at position +1 in LEGs and at position +2 in HEGs do not survive Bonferroni correction. For all other positions, there was no significant difference in any expression group (p > 0.05, Kruskal-Wallis).
Fig 4
Fig 4. Assessment of fourth base nucleotide frequencies as a function of primary stop usage.
Standard errors represent bootstrapped standard error. In all genes, not only is +4T enriched, compared to the next highest base, in TGA-terminating genes (T > A: p < 2.2 x 10−16, Wilcoxon signed-rank test), but consistent with the RF2 crosslinking hypothesis, the +4T usage was in the order TGA>TAA>TAG.
Fig 5
Fig 5. Enrichment of T-starting codons at position +1.
Enrichment scores calculated for each T-starting codon at position +1 such that: Enrichment Score = [F1 / mean (F3 + F4 + F5 + F6)] -1, where F1 = frequency at position +1 etc. Stop codons, highlighted in blue, show no remarkable enrichment compared to other T-starting codons.
Fig 6
Fig 6. ASC-containing genes (at position +N, where N is a downstream codon position from +1 to +5) compared against ASC-absent genes for the presence of another ASC at the next codon position.
Bars represent the median difference between ASC-absent and ASC-containing genes at each focal position. Error bars represent bootstrapped standard error. In the ‘all genes’ group, where an ASC is present at position +1 there is a significantly reduced chance of having another ASC at position +2 (Wilcoxon signed-rank test: p = 3.6 x 10−3).
Fig 7
Fig 7. Relative usage of each stop codon in 3’ UTRs plotted against GC3 content for TT11 bacterial species.
Surprisingly, we find that trends in the decoupled TGA and TAG to be consistent across all three reading frames. Spearman’s rank correlation information can be found in S5 Table.
Fig 8
Fig 8. Z-score analysis of previously analysed eukaryotic genomes.
Z-scores representing deviation from dinucleotide controlled null simulations over the whole 3’ UTR sequence (a) and then each position individually (b) for three eukaryotic genomes.
Fig 9
Fig 9
Number of genomes showing enrichment over dinucleotide-controlled null at each position, excluding position +1, in two genome sets (a) translation table 11 bacteria (n = 644) and (b) single-celled eukaryotes (n = 68). Genomes showing enrichment are underrepresented in the bacterial set (21/644, p = 0.028, one-tailed binomial test, expected = 32) and overrepresented in the eukaryotic set (21/68, p = 6.12 x 10−12, one tailed-binomial test, expected = 3). ‘Enrich’ is the total number of genomes with enrichment at one or more positions. ‘No enrich.’ is the total number of genomes with no enrichment at any position.
Fig 10
Fig 10
Number of genomes showing enrichment over A+C null (see methods) at each position, excluding position +1, in two genome sets (a) translation table 11 bacteria (n = 644) and (b) single-celled eukaryotes (n = 68). Genomes showing enrichment are underrepresented in the bacterial set (7/644, p = 9.5 x 10−8, one-tailed binomial test with p = 0.049, expected = 32) and overrepresented in the protists set (32/68, p = < 2.2 x 10−16, one tailed-binomial test with p = 0.049, expected = 3). ‘Enrich’ is the total number of genomes with enrichment at one or more positions. ‘No enrich.’ is the total number of genomes with no enrichment at any position.

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References

    1. Warnecke T, Hurst LD. Error prevention and mitigation as forces in the evolution of genes and genomes. Nat Rev Genet. 2011;12(12):875–81. 10.1038/nrg3092 WOS:000297252500013. - DOI - PubMed
    1. Fu Q, Liu CJ, Zhang X, Zhai ZS, Wang YZ, Hu MX, et al. Glucocorticoid receptor regulates expression of microRNA-22 and downstream signaling pathway in apoptosis of pancreatic acinar cells. World Journal of Gastroenterology. 2018;24(45):5120–30. 10.3748/wjg.v24.i45.5120 WOS:000452759500007. - DOI - PMC - PubMed
    1. Liu Z, Zhang JZ. Human C-to-U coding RNA editing is largely nonadaptive. Mol Biol Evol. 2018;35(4):963–9. 10.1093/molbev/msy011 WOS:000431889000014. - DOI - PMC - PubMed
    1. Liu Z, Zhang JZ. Most m(6)A RNA modifications in protein-coding regions are evolutionarily unconserved and likely nonfunctional. Mol Biol Evol. 2018;35(3):666–75. 10.1093/molbev/msx320 WOS:000427260700013. - DOI - PMC - PubMed
    1. Yang JR, Maclean CJ, Park C, Zhao HB, Zhang JZ. Intra and interspecific variations of gene expression levels in yeast are largely neutral: (Nei Lecture, SMBE 2016, Gold Coast). Mol Biol Evol. 2017;34(9):2125–39. 10.1093/molbev/msx171 WOS:000408307400001. - DOI - PMC - PubMed

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Grant support

This work was supported by the European Research Council (Grant EvoGenMed ERC-2014-ADG 669207 to L.D.H). For more information regarding ERC activities, please visit https://erc.europa.eu/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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