Fitness landscape of a dynamic RNA structure

Abstract

RNA structures are dynamic. As a consequence, mutational effects can be hard to rationalize with reference to a single static native structure. We reasoned that deep mutational scanning experiments, which couple molecular function to fitness, should capture mutational effects across multiple conformational states simultaneously. Here, we provide a proof-of-principle that this is indeed the case, using the self-splicing group I intron from Tetrahymena thermophila as a model system. We comprehensively mutagenized two 4-bp segments of the intron that come together to form the P1 extension (P1ex) helix at the 5 splice site and, following cleavage at the 5 splice site, dissociate to allow formation of an alternative helix (P10) at the 3 splice site. Using an in vivo reporter system that couples splicing activity to fitness in E. coli, we demonstrate that fitness is driven jointly by constraints on P1ex and P10 formation and that patterns of epistasis can be used to infer the presence of intramolecular pleiotropy. Importantly, using a machine learning approach that allows quantification of mutational effects in a genotype-specific manner, we show that the fitness landscape can be deconvoluted to implicate P1ex or P10 as the effective genetic background in which molecular fitness is compromised or enhanced. Our results highlight deep mutational scanning as a tool to study transient but important conformational states, with the capacity to provide critical insights into the evolution and evolvability of RNAs as dynamic ensembles. Our findings also suggest that, in the future, deep mutational scanning approaches might help us to reverse-engineer dynamic interactions and critical non-native states from a single fitness landscape.