Post-transcriptional mechanisms modulate the consequences of adaptive copy number variation.

Copy-number variants (CNVs) are large-scale amplifications or deletions of DNA that can drive rapid adaptive evolution and result in large-scale changes in gene expression. Whereas alterations in the copy number of one or more genes within a CNV can confer a selective advantage, other genes within a CNV can decrease fitness when their dosage is changed. Dosage compensation - in which the gene expression output from multiple gene copies is less than expected - is one means by which an organism can mitigate the fitness costs of deleterious gene amplification. Previous research has shown evidence for dosage compensation at both the transcriptional level and at the level of protein expression; however, the extent of compensation differs substantially between genes, strains, and studies. Here, we investigated sources of dosage compensation at multiple levels of gene expression regulation by defining the transcriptome, translatome and proteome of experimentally evolved yeast (Saccharomyces cerevisiae) strains containing adaptive CNVs. We quantified the gene expression output at each step and found evidence of widespread dosage compensation at the protein abundance (~47%) level. By contrast we find only limited evidence for dosage compensation at the transcriptional (~8%) and translational (~3%) level. We also find substantial divergence in the expression of unamplified genes in evolved strains that could be due to either the presence of a CNV or adaptation to the environment. Detailed analysis of 82 amplified and 411 unamplified genes with significantly discrepant relationships between RNA and protein abundances identified enrichment for upstream open reading frames (uORFs). These uORFs are enriched for binding site motifs for SSD1, an RNA binding protein that has previously been associated with tolerance of aneuploidy. Our findings suggest that, in the presence of CNVs, SSD1 may act to alter the expression of specific genes by potentiating uORF mediated translational regulation.

Best Practices in Microbial Experimental Evolution: Using Reporters and Long-Read Sequencing to Identify Copy Number Variation in Experimental Evolution.

Copy number variants (CNVs), comprising gene amplifications and deletions, are a pervasive class of heritable variation. CNVs play a key role in rapid adaptation in both natural, and experimental, evolution. However, despite the advent of new DNA sequencing technologies, detection and quantification of CNVs in heterogeneous populations has remained challenging. Here, we summarize recent advances in the use of CNV reporters that provide a facile means of quantifying de novo CNVs at a specific locus in the genome, and nanopore sequencing, for resolving the often complex structures of CNVs. We provide guidance for the engineering and analysis of CNV reporters and practical guidelines for single-cell analysis of CNVs using flow cytometry. We summarize recent advances in nanopore sequencing, discuss the utility of this technology, and provide guidance for the bioinformatic analysis of these data to define the molecular structure of CNVs. The combination of reporter systems for tracking and isolating CNV lineages and long-read DNA sequencing for characterizing CNV structures enables unprecedented resolution of the mechanisms by which CNVs are generated and their evolutionary dynamics.