Jian Rong Yang

Balanced codon usage optimizes eukaryotic translational efficiency.

Cellular efficiency in protein translation is an important fitness determinant in rapidly growing organisms. It is widely believed that synonymous codons are translated with unequal speeds and that translational efficiency is maximized by the exclusive use of rapidly translated codons. Here we estimate the in vivo translational speeds of all sense codons from the budding yeast Saccharomyces cerevisiae. Surprisingly, preferentially used codons are not translated faster than unpreferred ones. We hypothesize that this phenomenon is a result of codon usage in proportion to cognate tRNA concentrations, the optimal strategy in enhancing translational efficiency under tRNA shortage. Our predicted codon–tRNA balance is indeed observed from all model eukaryotes examined, and its impact on translational efficiency is further validated experimentally. Our study reveals a previously unsuspected mechanism by which unequal codon usage increases translational efficiency, demonstrates widespread natural selection for translational efficiency, and offers new strategies to improve synthetic biology.

Protein misinteraction avoidance causes highly expressed proteins to evolve slowly

The tempo and mode of protein evolution have been central questions in biology. Genomic data have shown a strong influence of the expression level of a protein on its rate of sequence evolution (E-R anticorrelation), which is currently explained by the protein misfolding avoidance hypothesis. Here, we show that this hypothesis does not fully explain the E-R anticorrelation, especially for protein surface residues. We propose that natural selection against protein–protein misinteraction, which wastes functional molecules and is potentially toxic, constrains the evolution of surface residues. Because highly expressed proteins are under stronger pressures to avoid misinteraction, surface residues are expected to show an E-R anticorrelation. Our molecular-level evolutionary simulation and yeast genomic analysis confirm multiple predictions of the hypothesis. These findings show a pluralistic origin of the E-R anticorrelation and reveal the role of protein misinteraction, an inherent property of complex cellular systems, in constraining protein evolution.

Determinants of the rate of protein sequence evolution

The rate and mechanism of protein sequence evolution have been central questions in evolutionary biology since the 1960s. Although the rate of protein sequence evolution depends primarily on the level of functional constraint, exactly what determines functional constraint has remained unclear. The increasing availability of genomic data has enabled much needed empirical examinations on the nature of functional constraint. These studies found that the evolutionary rate of a protein is predominantly influenced by its expression level rather than functional importance. A combination of theoretical and empirical analyses has identified multiple mechanisms behind these observations and demonstrated a prominent role in protein evolution of selection against errors in molecular and cellular processes.

Pseudogenization of the Umami Taste Receptor Gene Tas1r1 in the Giant Panda Coincided with its Dietary Switch to Bamboo

Although it belongs to the order Carnivora, the giant panda is a vegetarian with 99% of its diet being bamboo. The draft genome sequence of the giant panda shows that its umami taste receptor gene Tas1r1 is a pseudogene, prompting the proposal that the loss of the umami perception explains why the giant panda is herbivorous. To test this hypothesis, we sequenced all six exons of Tas1r1 in another individual of the giant panda and five other carnivores. We found that the open reading frame (ORF) of Tas1r1 is intact in all these carnivores except the giant panda. The rate ratio (ω) of nonsynonymous to synonymous substitutions in Tas1r1 is significantly higher for the giant panda lineage than for other carnivore lineages. Based on the ω change and the observed number of ORF-disrupting substitutions, we estimated that the functional constraint on the giant panda Tas1r1 was relaxed ∼ 4.2 Ma, with its 95% confidence interval between 1.3 and 10 Ma. Our estimate matches the approximate date of the giant pandas dietary switch inferred from fossil records. It is probable that the giant pandas decreased reliance on meat resulted in the dispensability of the umami taste, leading to Tas1r1 pseudogenization, which in turn reinforced its herbivorous life style because of the diminished attraction of returning to meat eating in the absence of Tas1r1. Nonetheless, additional factors are likely involved because herbivores such as cow and horse still retain an intact Tas1r1.

Impact of translational error-induced and error-free misfolding on the rate of protein evolution

What determines the rate of protein evolution is a fundamental question in biology. Recent genomic studies revealed a surprisingly strong anticorrelation between the expression level of a protein and its rate of sequence evolution. This observation is currently explained by the translational robustness hypothesis in which the toxicity of translational error‐induced protein misfolding selects for higher translational robustness of more abundant proteins, which constrains sequence evolution. However, the impact of error‐free protein misfolding has not been evaluated. We estimate that a non‐negligible fraction of misfolded proteins are error free and demonstrate by a molecular‐level evolutionary simulation that selection against protein misfolding results in a greater reduction of error‐free misfolding than error‐induced misfolding. Thus, an overarching protein‐misfolding‐avoidance hypothesis that includes both sources of misfolding is superior to the translational robustness hypothesis. We show that misfolding‐minimizing amino acids are preferentially used in highly abundant yeast proteins and that these residues are evolutionarily more conserved than other residues of the same proteins. These findings provide unambiguous support to the role of protein‐misfolding‐avoidance in determining the rate of protein sequence evolution.

Differential requirements for mRNA folding partially explain why highly expressed proteins evolve slowly

Significance The expression level of a gene is a leading determinant of its rate of protein sequence evolution, but the underlying mechanisms are unclear. We show that as the mRNA concentration increases, natural selection for mRNA folding intensifies, resulting in larger fractions of mutations deleterious to mRNA folding and lower rates of protein evolution. Counterintuitively, selection for mRNA folding also impacts the nonsynonymous-to-synonymous nucleotide substitution rate ratio, requiring a revision of the current interpretation of this ratio as a measure of protein-level selection. These findings demonstrate a prominent role of selection at the mRNA level in molecular evolution. The cause of the tremendous among-protein variation in the rate of sequence evolution is a central subject of molecular evolution. Expression level has been identified as a leading determinant of this variation among genes encoded in the same genome, but the underlying mechanisms are not fully understood. We here propose and demonstrate that a requirement for stronger folding of more abundant mRNAs results in slower evolution of more highly expressed genes and proteins. Specifically, we show that: (i) the higher the expression level of a gene, the greater the selective pressure for its mRNA to fold; (ii) random mutations are more likely to decrease mRNA folding when occurring in highly expressed genes than in lowly expressed genes; and (iii) amino acid substitution rate is negatively correlated with mRNA folding strength, with or without the control of expression level. Furthermore, synonymous (dS) and nonsynonymous (dN) nucleotide substitution rates are both negatively correlated with mRNA folding strength. However, counterintuitively, dS and dN are differentially constrained by selection for mRNA folding, resulting in a significant correlation between mRNA folding strength and dN/dS, even when gene expression level is controlled. The direction and magnitude of this correlation is determined primarily by the G+C frequency at third codon positions. Together, these findings explain why highly expressed genes evolve slowly, demonstrate a major role of natural selection at the mRNA level in constraining protein evolution, and reveal a previously unrecognized and unexpected form of nonprotein-level selection that impacts dN/dS.

Codon-by-Codon Modulation of Translational Speed and Accuracy Via mRNA Folding

Secondary structure in mRNAs modulates the speed of protein synthesis codon-by-codon to improve accuracy at important sites while ensuring high speed elsewhere.

Human Long Noncoding RNAs Are Substantially Less Folded than Messenger RNAs

Long noncoding RNAs (lncRNAs) do not code for proteins but function as RNAs. Because the functions of an RNA rely on either its sequence or secondary structure, lncRNAs should be folded at least as strongly as messenger RNAs (mRNAs), which serve as messengers for translation and are generally thought to lack secondary structure-dependent RNA-level functions. Contrary to this prediction, analysis of genome-wide experimental data of human RNA folding reveals that lncRNAs are substantially less folded than mRNAs even after the control of expression level and GC% (percentage of guanines and cytosines), although both lncRNAs and mRNAs are more strongly folded than expected by chance. In contrast to mRNAs, lncRNAs show neither the positive correlation between folding strength and expression level nor the negative correlation between folding strength and evolutionary rate. These and other results support that although RNA folding undoubtedly plays a role in RNA biology it is also important in translation and/or protein biology.

Nascent RNA folding mitigates transcription-associated mutagenesis

Transcription is mutagenic, in part because the R-loop formed by the binding of the nascent RNA with its DNA template exposes the nontemplate DNA strand to mutagens and primes unscheduled error-prone DNA synthesis. We hypothesize that strong folding of nascent RNA weakens R-loops and hence decreases mutagenesis. By a yeast forward mutation assay, we show that strengthening RNA folding and reducing R-loop formation by synonymous changes in a reporter gene can lower mutation rate by >80%. This effect is diminished after the overexpression of the gene encoding RNase H1 that degrades the RNA in a DNA-RNA hybrid, indicating that the effect is R-loop-dependent. Analysis of genomic data of yeast mutation accumulation lines and human neutral polymorphisms confirms the generality of these findings. This mechanism for local protection of genome integrity is of special importance to highly expressed genes because of their frequent transcription and strong RNA folding, the latter also improves translational fidelity. As a result, strengthening RNA folding simultaneously curtails genotypic and phenotypic mutations.

Deciphering the Genic Basis of Yeast Fitness Variation by Simultaneous Forward and Reverse Genetics

The budding yeast Saccharomyces cerevisiae is the best studied eukaryote in molecular and cell biology, but its utility for understanding the genetic basis of phenotypic variation in natural populations is limited by inefficient association mapping due to strong and complex population structure. To overcome this challenge, we generated genome sequences for 85 strains and performed a comprehensive population genomic survey of a total of 190 diverse strains. We identified considerable variation in population structure among chromosomes and identified 181 genes that are absent from the reference genome. Many of these nonreference genes are expressed and we functionally confirmed that two of these genes confer increased resistance to antifungals. Next, we simultaneously measured the growth rates of over 4,500 laboratory strains, each of which lacks a nonessential gene, and 81 natural strains across multiple environments using unique DNA barcode present in each strain. By combining the genome-wide reverse genetic information gained from the gene deletion strains with a genome-wide association analysis from the natural strains, we identified genomic regions associated with fitness variation in natural populations. To experimentally validate a subset of these associations, we used reciprocal hemizygosity tests, finding that while the combined forward and reverse genetic approaches can identify a single causal gene, the phenotypic consequences of natural genetic variation often follow a complicated pattern. The resources and approach provided outline an efficient and reliable route to association mapping in yeast and significantly enhance its value as a model for understanding the genetic mechanisms underlying phenotypic variation and evolution in natural populations.