Pervasive RNA Folding is Crucial for Narnavirus Genome Maintenance.

A synthetic biology approach toward constructing an RNA -based genome expands our understanding of living things and opens avenues for technological advancement. For the precise design of an artificial RNA replicon either from scratch or based on a natural RNA replicon, understanding structure-function relationships of RNA sequences is critical. However, our knowledge remains limited to a few particular structural elements intensively studied so far. Here, we conducted a series of site-directed mutagenesis studies of yeast narnaviruses ScNV20S and ScNV23S, perhaps the simplest natural autonomous RNA replicons, to identify RNA elements required for maintenance and replication. RNA structure disruption corresponding to various portions of the entire narnavirus genome suggests that pervasive RNA folding, in addition to the precise sec-ondary structure of genome termini, is essential for maintenance of the RNA replicon in vivo. Computational RNA structure analyses suggest that this scenario likely applies to other "narna-like" viruses. This finding implies selective pressure on these simplest autonomous natural RNA replicons to fold into a unique structure that acquires both thermodynamic and biological stability. We propose the importance of pervasive RNA folding for the design of RNA replicons that could serve as a platform for in vivo con-tinuous evolution as well as an interesting model to study the origin of life.SignificanceArtificial RNA replicons could serve as a platform to study the origin of life as well as to promote new technological advancement. To engineer an RNA replicon, understanding the structure-function relationship of the sequence is critical. To gain a deeper insight into it, we focused on the virus family Narnaviridae that have a simple single -stranded RNA genome. By using a combined approach of computational RNA structure analysis and in vivo examination of each structural element, we found that not only typical terminal RNA structures but also pervasive internal RNA folding of the replicon is critical for the maintenance of the RNA in vivo. This finding implies that taking account of pervasive folding status is important for RNA replicon design.