Geneticists and evolutionary biologists have for decades embraced the view, no doubt reinforced by terminology such as “silent” (i.e., synonymous) mutations, that for protein-encoding genes, genotype determines phenotype through control of the amino acid sequence of the corresponding polypeptide chain or chains. In various contexts, the standard assumption has therefore been that only non-synonymous nucleotide substitutions in a gene have an impact on the phenotype and are subject to selection.
In 2007, Kimchy-Sarfaty et al. (Science) described a likely exception to the conventional wisdom outlined above in a study of the Multidrug Resistance 1 (MDR1) gene product, P-glycoprotein, or P-gp. They found that a particular synonymous mutation in the MDR1 gene, C3435T, when found in conjunction with one or two other mutations, one synonymous and one non-synonymous, was associated with altered P-gp function. The P-gp encoded by the multiply mutated gene differed from the P-gp encoded by the wild-type gene in ability to mediate the efflux of various fluorescent drugs, in the presence of some but not other inhibitors, from any of several different human and monkey cell lines. None of the P-gp proteins bearing single mutations exhibited altered function. Since the function differences between wild-type and the multiply-mutated P-gp increased with increasing mRNA levels (as controlled by varying the amount of DNA used for transfection of the cell lines), the authors speculated that differences in cotranslational folding of the P-gp polypeptide might account for the functional results as well as for evidence that the wild-type and multiply-mutated P-gp proteins exhibited differences in conformation.
About one year ago, Coleman et al. (Science, 2008) showed that poliovirus could be attenuated by introducing over 600 synonymous mutations, in the form of unusual (relative to the genomic DNA of humans) pairs of adjacent codons, in the P1 region of the viral genome, which encodes the capsid. This codon pair bias caused the rate of translation for poliovirus gene products to decrease significantly. Infection of mice with the attenuated virus effectively protected against subsequent challenge with an otherwise lethal dose of wild-type virus that killed all mock-immunized mice.
Another relatively recent report that challenges the conventional wisdom on silent mutations is from Joshua Plotkin and colleagues at the University of Pennsylvania (Science, 2008). They show that variants of green fluorescent protein carrying randomly distributed synonymous mutations varied in expression level, in E. coli, over a 250-fold range. More than half of the variation in magnitude of gene expression could be accounted for by differences in mRNA folding and correlated differences in the rate of translation initiation. Somewhat unexpectedly, codon bias did not appear to correlate with gene expression, but it did correlate with the rate of cellular growth.
In an informative and interesting recent summary of phenotypic consequences of synonymous mutations, Chamary and Hurst (Sci Am. 2009) identify a number of mechanisms that can account for such effects. These mechanisms include:
Effects on rate of protein synthesis (due to limiting quantities of tRNA’s for non-preferred codons, or due to alterations in splicing enhancers that alter splicing efficiency)
Effects on accuracy of protein synthesis (due to variable recognition of different synonymous codons by particular tRNAs)
Synthesis-rate-dependent effects on protein folding, leading to altered protein function
According to these latter authors, as many as 50 genetic disorders have now been attributed to synonymous mutations, many involving altered splicing of mRNAs.
Kimchi-Sarfaty C, Oh JM, Kim IW, Sauna ZE, Calcagno AM, Ambudkar SV, Gottesman MM. A “silent” polymorphism in the MDR1 gene changes substrate specificity. Science. 2007 Jan 26;315(5811):525-8. Epub 2006 Dec 21. Erratum in: Science. 2007 Nov 30;318(5855):1382-3.
Coleman JR, Papamichail D, Skiena S, Futcher B, Wimmer E, Mueller S. Virus attenuation by genome-scale changes in codon pair bias. Science. 2008 Jun 27;320(5884):1784-7.
Kudla G, Murray AW, Tollervey D, Plotkin JB. Coding-sequence determinants of gene expression in Escherichia coli. Science. 2009 Apr 10;324(5924):255-8.
Chamary JV, Hurst LD. The price of silent mutations. Sci Am. 2009 Jun;300(6):46-53.