This guest post is by Carlo Arteri and Hunter Fraser on their preprint Evolution at two levels of gene expression in yeast, arXived here

Taking studies of regulatory evolution to the next level: translation

Understanding the molecular basis of regulatory variation within and between species has become a major focus of modern genetics. For instance, the majority of identified human disease-risk alleles lie in non-coding regions of the genome, suggesting that they affect gene regulation (Epstein 2009). Furthermore, it has been argued that regulatory changes have played a dominant role in explaining uniquely human attributes (King and Wilson 1975). However, our knowledge of gene regulatory evolution is based almost entirely on studies of mRNA levels, despite both the greater functional importance of protein abundance, and evidence that post-transcriptional regulation is pervasive. The availability of high-throughput methods for measuring mRNA abundance coupled to the lack of comparable methods at the protein level have contributed to this focus; however, a new method known as ribosome profiling (Ingolia et al. 2009) has enabled us to study divergence in the regulation of translation.

‘Riboprofiling’ involves the construction of two RNA-seq libraries: one measuring mRNA abundance (the ‘mRNA’ fraction), and the second capturing the portion of the transcriptome that is actively being translated by ribosomes (the ‘Ribo’ fraction). We performed riboprofiling on interspecific hybrids of two closely related species of budding yeast, Saccharomyces cerevisiae and S. paradoxus, (~5 million years diverged) as well as the parental strains. As both parental alleles at a locus share the same trans cellular environment in the hybrid, differences in the relative allelic abundance (termed allele-specific expression, or ASE) reveal cis-regulatory divergence. Consequently, interspecies differences not attributable to cis-effects indicate trans divergence. By measuring differences in the magnitudes of ASE between the two hybrid riboprofiling fractions, we identified independent cis and trans regulatory changes in both mRNA abundance and translational efficiency.

We found that both cis and trans regulatory divergence in translation are widespread, and of comparable magnitude to divergence at the mRNA level – indicating that we miss much regulatory evolution by focusing on mRNA in isolation. Moreover, we observed an overwhelming bias towards divergence in opposing parental directions, suggesting the action of stabilizing selection in order to maintain more similar protein levels between species than would be expected by comparing mRNA abundances alone. Interestingly, while we confirmed the results of previous studies indicating that both cis and trans regulatory divergence at the mRNA level are associated with the presence of TATA boxes and nucleosome free regions in promoters, no such relationship was found for translational divergence, indicating that these regulatory systems have different underlying architectures.

We also searched for evidence of polygenic selection in and between both regulatory levels by applying a recently developed modification of Orr’s sign test (Orr 1998; Fraser et al. 2010; Bullard et al. 2010). Under neutral divergence, no pattern is expected with regards to the parental direction of up or down-regulating alleles among orthologs within a functional group (e.g., a pathway or multi-gene complex). However, a significant bias towards one parental lineage is evidence of lineage-specific selection. This analysis uncovered evidence of polygenic selection at both regulatory levels in a number of functional groups. In particular, genes involved in tolerance to heavy metals were enriched for reinforcing divergence in mRNA abundance and translation favoring S. cerevisiae. Increased tolerance to these metals has been observed in S. cerevisiae (Warringer et al. 2011), suggesting that domesticated yeasts have experienced a history of polygenic adaptation across regulatory levels allowing them to grow on metals such as copper. Finally, we also uncovered multiple instances of stop-codon readthrough that are conserved between species, highlighting yet another post-transcriptional mechanism leading to increased proteomic diversity.

By applying a novel approach to a long-standing question, our analysis has revealed the underappreciated complexity of post-transcriptional regulatory divergence. We argue that partitioning the search for the locus of selection into the binary categories of ‘coding’ vs. ‘regulatory’ overlooks the many opportunities for selection to act at multiple regulatory levels along the path from genotype to phenotype.

References:

Bullard JH, Mostovoy Y, Dudoit S, Brem RB. 2010. Polygenic and directional regulatory evolution across pathways in Saccharomyces. Proc Natl Acad Sci USA 107: 5058-5063.

Epstein DJ. 2009. Cis-regulatory mutations in human disease. Brief Funct Genomic Proteomic 8: 310–316.

Fraser HB, Moses AM, Schadt EE. 2010. Evidence for widespread adaptive evolution of gene expression in budding yeast. Proc Natl Acad Sci USA 107: 2977-2982.

Ingolia NT, Ghaemmaghami S, Newman JR, Weissman JS. 2009. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324:218-223.

King MC, Wilson AC. 1975. Evolution at two levels in humans and chimpanzees. Science 188: 107-116.

Orr HA. 1998. Testing natural selection vs. genetic drift in phenotypic evolution using quantitative trait locus data. Genetics 149: 2099-2104.

Warringer J, Zörgö E, Cubillos FA, Zia A, Gjuvsland A, Simpson JT, Forsmark A, Durbin R, Omholt SW, Louis EJ, Liti G, Moses A, Blomberg A. 2011. Trait variation in yeast is defined by population history. PLoS Genet 7 :e1002111.