Intramolecular phenotypic capacitance in a modular RNA molecule

Abstract

Phenotypic capacitance refers to the ability of a genome to accumulate mutations that are conditionally hidden and only reveal phenotype-altering effects after certain environmental or genetic changes. Capacitance has important implications for the evolution of novel forms and functions, but experimentally studied mechanisms behind capacitance are mostly limited to complex, multicomponent systems often involving several interacting protein molecules. Here we demonstrate phenotypic capacitance within a much simpler system, an individual RNA molecule with catalytic activity (ribozyme). This naturally occurring RNA molecule has a modular structure, where a scaffold module acts as an intramolecular chaperone that facilitates folding of a second catalytic module. Previous studies have shown that the scaffold module is not absolutely required for activity, but dramatically decreases the concentration of magnesium ions required for the formation of an active site. Here, we use an experimental perturbation of magnesium ion concentration that disrupts the folding of certain genetic variants of this ribozyme and use in vitro selection followed by deep sequencing to identify genotypes with altered phenotypes (catalytic activity). We identify multiple conditional mutations that alter the wild-type ribozyme phenotype under a stressful environmental condition of low magnesium ion concentration, but preserve the phenotype under more relaxed conditions. This conditional buffering is confined to the scaffold module, but controls the catalytic phenotype, demonstrating how modularity can enable phenotypic capacitance within a single macromolecule. RNA's ancient role in life suggests that phenotypic capacitance may have influenced evolution since life's origins. modularity | epistasis | phenotypic capacitance | ribozyme | evolution

Empirical analysis of RNA robustness and evolution using high-throughput sequencing of ribozyme reactions

“…Deleterious mutation effects are buffered by high magnesium concentrations, but revealed at low magnesium concentrations. This data was published as supporting information in a previous publication [14]. Figure 3.…”

“…Fitness effects and interactions are equally important at the level of individual genes [25]. However, the fitness landscapes of proteins and RNA molecules encountered by periods of mutation without selection remain poorly understood [14,26]. Random mutation accumulation may be a critical source of new functions following gene duplication.…”

“…We previously carried out random mutation addition in the Azoarcus ribozyme [14]. To accomplish this, we used error-prone PCR to introduce mutations at a rate of about one mutation per individual per PCR (see Methods).…”

“…More specifically, they can guide our understanding of RNA evolution in the laboratory and in natural systems. This article reviews several recent empirical approaches to ribozyme evolution that were aimed at 5 understanding how mutational robustness, standing genetic variation and environmental change can promote adaptation [13][14][15].…”

Information theory and the phenotypic complexity of evolutionary adaptations and innovations

“…The first is in vitro selection [33]. Here, large collections ("libraries") of DNA or RNA genotypes are synthesized, and molecules with specific phenotypes, such as the ability to bind ATP, are selected via methods like affinity chromatography [34][35][36][37]. A variant of this approach is found in protein-binding microarrays, where a library of DNA molecules is immobilized on an array.…”