Vladimir N. Babenko

Evolutionary conservation suggests a regulatory function of AUG triplets in 5′-UTRs of eukaryotic genes

By comparing sequences of human, mouse and rat orthologous genes, we show that in 5′-untranslated regions (5′-UTRs) of mammalian cDNAs but not in 3′-UTRs or coding sequences, AUG is conserved to a significantly greater extent than any of the other 63 nt triplets. This effect is likely to reflect, primarily, bona fide evolutionary conservation, rather than cDNA annotation artifacts, because the excess of conserved upstream AUGs (uAUGs) is seen in 5′-UTRs containing stop codons in-frame with the start AUG and many of the conserved AUGs are found in different frames, consistent with the location in authentic non-coding sequences. Altogether, conserved uAUGs are present in at least 20–30% of mammalian genes. Qualitatively similar results were obtained by comparison of orthologous genes from different species of the yeast genus Saccharomyces. Together with the observation that mammalian and yeast 5′-UTRs are significantly depleted in overall AUG content, these findings suggest that AUG triplets in 5′-UTRs are subject to the pressure of purifying selection in two opposite directions: the uAUGs that have no specific function tend to be deleterious and get eliminated during evolution, whereas those uAUGs that do serve a function are conserved. Most probably, the principal role of the conserved uAUGs is attenuation of translation at the initiation stage, which is often additionally regulated by alternative splicing in the mammalian 5′-UTRs. Consistent with this hypothesis, we found that open reading frames starting from conserved uAUGs are significantly shorter than those starting from non-conserved uAUGs, possibly, owing to selection for optimization of the level of attenuation.

Reconstruction of Ancestral Protosplice Sites

Most of the eukaryotic protein-coding genes are interrupted by multiple introns. A substantial fraction of introns occupy the same position in orthologous genes from distant eukaryotes, such as plants and animals, and consequently are inferred to have been inherited from the common ancestor of these organisms. In contrast to these conserved introns, many other introns appear to have been gained during evolution of each major eukaryotic lineage. The mechanism(s) of insertion of new introns into genes remains unknown. Because the nucleotides that flank splice junctions are nonrandom, it has been proposed that introns are preferentially inserted into specific target sequences termed protosplice sites. However, it remains unclear whether the consensus nucleotides flanking the splice junctions are remnants of the original protosplice sites or if they evolved convergently after intron insertion. Here, we directly address the existence of protosplice sites by examining the context of introns inserted within codons that encode amino acids conserved in all eukaryotes and accordingly are not subject to selection for splicing efficiency. We show that introns are either predominantly inserted into specific protosplice sites, which have the consensus sequence (A/C)AG/Gt, or that they are inserted randomly but are preferentially fixed at such sites.

Evidence of Splice Signal Migration from Exon to Intron during Intron Evolution

A comparison of the nucleotide sequences around the splice junctions that flank old (shared by two or more major lineages of eukaryotes) and new (lineage-specific) introns in eukaryotic genes reveals substantial differences in the distribution of information between introns and exons. Old introns have a lower information content in the exon regions adjacent to the splice sites than new introns but have a corresponding higher information content in the intron itself. This suggests that introns insert into nonrandom (proto-splice) sites but, during the evolution of an intron after insertion, the splice signal shifts from the flanking exon regions to the ends of the intron itself. Accumulation of information inside the intron during evolution suggests that new introns largely emerge de novo rather than through propagation and migration of old introns.

Signs of positive selection of somatic mutations in human cancers detected by EST sequence analysis

BackgroundCarcinogenesis typically involves multiple somatic mutations in caretaker (DNA repair) and gatekeeper (tumor suppressors and oncogenes) genes. Analysis of mutation spectra of the tumor suppressor that is most commonly mutated in human cancers, p53, unexpectedly suggested that somatic evolution of the p53 gene during tumorigenesis is dominated by positive selection for gain of function. This conclusion is supported by accumulating experimental evidence of evolution of new functions of p53 in tumors. These findings prompted a genome-wide analysis of possible positive selection during tumor evolution.MethodsA comprehensive analysis of probable somatic mutations in the sequences of Expressed Sequence Tags (ESTs) from malignant tumors and normal tissues was performed in order to access the prevalence of positive selection in cancer evolution. For each EST, the numbers of synonymous and non-synonymous substitutions were calculated. In order to identify genes with a signature of positive selection in cancers, these numbers were compared to: i) expected numbers and ii) the numbers for the respective genes in the ESTs from normal tissues.ResultsWe identified 112 genes with a signature of positive selection in cancers, i.e., a significantly elevated ratio of non-synonymous to synonymous substitutions, in tumors as compared to 37 such genes in an approximately equal-sized EST collection from normal tissues. A substantial fraction of the tumor-specific positive-selection candidates have experimentally demonstrated or strongly predicted links to cancer.ConclusionThe results of EST analysis should be interpreted with extreme caution given the noise introduced by sequencing errors and undetected polymorphisms. Furthermore, an inherent limitation of EST analysis is that multiple mutations amenable to statistical analysis can be detected only in relatively highly expressed genes. Nevertheless, the present results suggest that positive selection might affect a substantial number of genes during tumorigenic somatic evolution.

Mutational hotspots in the TP53 gene and, possibly, other tumor suppressors evolve by positive selection.

BackgroundThe mutation spectra of the TP53 gene and other tumor suppressors contain multiple hotspots, i.e., sites of non-random, frequent mutation in tumors and/or the germline. The origin of the hotspots remains unclear, the general view being that they represent highly mutable nucleotide contexts which likely reflect effects of different endogenous and exogenous factors shaping the mutation process in specific tissues. The origin of hotspots is of major importance because it has been suggested that mutable contexts could be used to infer mechanisms of mutagenesis contributing to tumorigenesis.ResultsHere we apply three independent tests, accounting for non-uniform base compositions in synonymous and non-synonymous sites, to test whether the hotspots emerge via selection or due to mutational bias. All three tests consistently indicate that the hotspots in the TP53 gene evolve, primarily, via positive selection. The results were robust to the elimination of the highly mutable CpG dinucleotides. By contrast, only one, the least conservative test reveals the signature of positive selection in BRCA1, BRCA2, and p16. Elucidation of the origin of the hotspots in these genes requires more data on somatic mutations in tumors.ConclusionThe results of this analysis seem to indicate that positive selection for gain-of-function in tumor suppressor genes is an important aspect of tumorigenesis, blurring the distinction between tumor suppressors and oncogenes.ReviewersThis article was reviewed by Sandor Pongor, Christopher Lee and Mikhail Blagosklonny.

Purifying and positive selection in the evolution of stop codons

Modes of evolution of stop codons in protein-coding genes, especially the conservation of UAA, have been debated for many years. We reconstructed the evolution of stop codons in 40 groups of closely related prokaryotic and eukaryotic genomes. The results indicate that the UAA codons are maintained by purifying selection in all domains of life. In contrast, positive selection appears to drive switches from UAG to other stop codons in prokaryotes but not in eukaryotes. Changes in stop codons are significantly associated with increased substitution frequency immediately downstream of the stop. These positions are otherwise more strongly conserved in evolution compared to sites farther downstream, suggesting that such substitutions are compensatory. Although GC content has a major impact on stop codon frequencies, its contribution to the decreased frequency of UAA differs between bacteria and archaea, presumably, due to differences in their translation termination mechanisms.