So, does that mean it's time to throw out our idea that mutations in a gene that don't alter its protein sequence are neutral? And with it, all the tools we use to study protein evolution that are based on this assumption?
Worse than it looked —
Mutations thought to be harmless turn out to cause problems
Mutations in genes that don't alter proteins can still alter survival in yeast.
Selection enhances protein evolvability by increasing mutational robustness and foldability
Selection enhances mutation toleration
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But this week, a paper provided evidence that a significant category of mutations isn't as neutral as we thought they were. The big caveat is that the study was done in yeast, which is a weird organism in a couple of ways, so we'll have to see if the results hold in others. . .
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Make all the mutations
To test neutral mutations, the researchers started with a panel of 21 yeast genes, chosen partly because they are involved in a wide variety of cellular activities. The other part behind their choice is that eliminating these genes doesn't kill the yeast but makes it less healthy. That should make it easier to detect partial effects, where the mutation makes the yeast less healthy.
Within that stretch, the researchers picked a 150-base stretch in the DNA and made every single possible mutation, using DNA editing to make a yeast strain carrying the mutation. That is a total of over 9,000 individual yeast strains, with some carrying mutations that will change the amino acid sequence and others carrying mutations we'd expect to be neutral. But of course, this involved lab work, where things don't work for random, unknown reasons, so the researchers had to settle for testing about 8,300 mutant yeast strains.
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INSERT FYI Yeast Spontaneous Mutation Rate and Spectrum Vary with Environment
(Graphical Abstract: https://www.sciencedirect.com/science/article/pii/S0960982219303823)
The test was pretty simple. Throw equal numbers of normal and mutant yeast in a flask, and let them grow for a bit. Then, sample the population, and check the relative levels of normal and mutant yeast. If the mutation lowered the fitness, you'd see more normal yeast when you sampled the flask.
That was true for mutations that changed an amino acid.
These saw their relative fitness drop a bit, though not by much (their fitness was 0.988 that of the normal yeast). But the neutral mutations weren't notably different—they also dropped the yeast's fitness by a tiny amount relative to a normal strain. In effect, the mutations that didn't change any amino acids were, on average, indistinguishable from the ones that did. Beyond this average, you could tell a slight difference.
> There were more amino acid-altering mutations that had a stronger deleterious effect on fitness, and more neutral ones that had a minimal effect.
> But it's clear that, as a whole, the class expected to be neutral wasn't.
Wait, what?
On the surface, that doesn't make any sense. Both versions of the gene encode exactly the same amino acid. How could one possibly be less fit than the other?
The secret to understanding this is remembering that the gene's DNA isn't used directly to make a protein. Instead, it's transcribed into an RNA copy called a messenger RNA, and that is directly translated to make the protein. And alterations in the DNA can affect the RNA's three-dimensional structure, its stability in the cell, and the rate it's translated into protein.
> The researchers found that the mutations expected to be neutral often influenced the amount of messenger RNA present in the cell.
> They also appeared to influence the RNA's ability to fold into a three-dimensional shape.
> And they were also likely to affect the efficiency of the messenger RNA in translating into a protein.
Combined, these could account for why this group of mutations had a collective impact on fitness.
So, does that mean it's time to throw out our idea that mutations in a gene that don't alter its protein sequence are neutral? And with it, all the tools we use to study protein evolution that are based on this assumption?
The researchers give one major reason why this would be premature:
Yeasts are kind of weird.
To start, unlike animals, which mostly get a copy of a gene from their moms and another from their dads, yeast carries only one copy of every gene, so it will likely be more sensitive to subtle effects. Yeasts also live a lifestyle similar to bacteria, carrying a simplified genome and focusing on rapid reproduction—a relatively minor metabolic hit is more likely to slow them down.
And these effects are still very subtle. Even if you completely hammered the function of these proteins by creating a mutation that truncated the protein early, the fitness cost was slight (fitness was 0.94 of the yeast strain without any mutations). It's not even clear this behavior would occur in other genes in yeast, much less genes in other organisms.
The other thing that the researchers note is that, in actual populations that are evolving over the long term, evolutionary pressures are constantly shifting due to environmental changes. So, it could be that these mutations are effectively neutral in a realistic environment, so when we look at similar mutations in a natural population, they appear to be neutral.
All these results are an important caution and make it worth the time and effort needed to sort this out carefully."
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Why Should We Care About Neutral Evolution?
Neutral Theory: The Null Hypothesis of Molecular Evolution
In the decades since its introduction, the neutral theory of evolution has become central to the study of evolution at the molecular level, in part because it provides a way to make strong predictions that can be tested against actual data. The neutral theory holds that most variation at the molecular level does not affect fitness and, therefore, the evolutionary fate of genetic variation is best explained by stochastic processes. This theory also presents a framework for ongoing exploration of two areas of research: biased gene conversion, and the impact of effective population size on the effective neutrality of genetic variants.
The main goal of biology is to understand how living organisms function and how they adapt to ever-fluctuating environments. So one may wonder why it is important to study neutral evolutionary processes that, a priori, seem to have little effect on the evolution of phenotypes. There are actually three primary reasons for this adjusted focus.
First, as mentioned earlier, the neutral theory is the underlying basis of selection tests. These tests are widely used to identify functional elements (e.g., genes and regulatory regions) within genomic sequences. The basic principle of this comparative genomics approach is that functional elements are subject to selective pressure, and hence their pattern of evolution differs from the neutral expectation. To be able to detect selection, it is therefore necessary to have a good understanding of all the nonadaptive evolutionary processes that affect sequence evolution—mutation, BGC, and genetic drift. Notably, the selection test mentioned above requires knowledge of u. This parameter can be estimated by measuring K in sites that are expected, a priori, to be neutral—pseudogenes or defective transposable elements, for example—although u may vary across chromosomes. Also, in some eukaryotic taxa, BGC appears to have a strong influence on genome evolution by favoring the fixation of AT to GC mutations (Marias, 2003). This BGC drive leads to enrichment of GC-content in genomic regions that feature high crossover rates. Many lines of evidence indicate this process is responsible for the strong regional variations in GC-content across mammalian chromosomes. In mammals, crossovers occur essentially in hot spots (typically 1 to 2 kilobases long), and BGC can create strong substitution hot spots, where the local substitution rate can be up to 20 times higher than in the rest of the genome (Duret & Arndt, 2008). In some cases, BGC may even counteract the action of selection and lead to the fixation of deleterious mutations, which implies that BGC can contribute to species maladaptation. Thus, before concluding that sequences are subject to selection, it is necessary to test whether the observed pattern of sequence evolution cannot be explained by this nonadaptive process (Galtier & Duret, 2007).
A second reason why knowledge of neutral sequence evolution is important is that it provides information about molecular processes that are involved in genome functioning. For example, it has been found that in some taxa, there is an asymmetry of substitution patterns between the two DNA strands. This pattern is caused by the asymmetry of the DNA replication process and can be used to infer the location of replication origins within chromosomes (Lobry, 1996). Similarly, the asymmetry of substitution patterns can be used to detect and orient transcription in the germ line of mammals (Green et al., 2003). The analysis of neutral substitution patterns also revealed the existence of a homology-dependent mechanism of DNA methylation in primates (Meunier et al., 2005).
Finally, one point that is often not fully appreciated is that neutral evolution can ultimately contribute to phenotypic evolution and to species adaptation. Kimura noted that many gene duplications may get fixed by random genetic drift, simply because they are not deleterious . Then, because of relaxation of selective pressures, one or both copies may accumulate mutations that otherwise would have been counterselected. Some of these mutants will turn out to be useful for the adaptation of organisms to their environment (Kimura, 1991). The idea that nonadaptive processes may have a major impact on the evolution of biological complexity has been largely developed by Michael Lynch (Lynch, 2006). Following duplication, the functions of the duplicates are initially redundant. Also, most gene products contribute to multiple aspects of an organism's phenotype. If one duplicate undergoes a mutation that knocks out part of its contribution to phenotype, it is released from purifying selection to maintain those functions. The reduction of negative selection efficiency allows a wider exploration of the space of possible genotypes, which may allow for improvements in remaining functions.
Also, regardless of the steps involved, the evolutionary trajectory between one genotype and another better-fit genotype may sometimes have to pass through a less-optimal genotype. Thus, a reduction of effective population size, which increases the effect of random drift, may allow the fixation of weakly deleterious mutations to pass through this less-optimal genotype, and hence can open a new evolutionary trajectory, possibly toward better-adapted genotypes.
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